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Sample Preparation

Before you start your project

All users must contact Dr. David Kakhniashvili, PMC Director, and discuss specific project details before submitting samples to the PMC. You must also read the Sample Preparation Basics SOP for the PMC.

Sample Preparation Basics
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The quality of prepared samples is the top priority!
The quality of prepared samples may be affected by:

  1. External contaminants
  2. Reagents used for sample preparation/processing
  3. Abundant sample proteins

External Contaminants

  1. Contaminants may be introduced at several steps during sample preparation. Some contaminants may ruin MS analysis. Investigators who do not follow these recommendations for sample preparation will be charged for MS analysis, even their requested experiment fails during LC/MS analysis.
  2. Keratins are the most common contaminating proteins. The presence of keratins or other contaminant proteins is a problem limiting identification of target proteins. Keratin sources include air, dust, skin, hair, clothes, some chemicals – keratins are everywhere, and on everything. Keep bench/working surface clean and samples covered or protected as much as possible.
  3. Serum proteins (BSA, immunoglobulins, etc.) are frequently observed contaminant proteins often originating from cell culture media (when cell pellets are not thoroughly washed with PBS). Thoroughly wash cell pellets with PBS before proteins are extracted.
  4. Glycerol, PEG and similar polymers severely affect LC/MS analysis, even if present at very low concentrations. In addition, polymers tend to stick to an HPLC column and may ruin it. Sources of these contaminants include soaps/detergents, hand-creams, some reagents and plastics.
  5. Wear powder-free gloves at all times during sample preparation. Do not touch contamination sources (see above) with outer working surface of gloves; if you come into contact, change gloves.
  6. Use new disposable tubes, pipettes, tips. Thoroughly wash/clean anything un-disposable that will come into contact with your sample (i.e. centrifugal tubes, gel apparatus, staining trays, gel excising implements, gel imaging or storage equipment, etc.). Cleaning is required to remove keratins and other contaminants.
  7. Do not use plastic or glassware previously exposed to washing detergents. Detergents interfere with LC/MS analysis.
  8. Use new molecular mass cut off filter devices; wash to remove filter preservatives before use.
  9. Tubes/vials/Parafilm may be a source of extractable contaminants detrimental to LC/MS analysis. Do not use colored tubes or coated (‘low-binding’) vials/tubes or vials with rubber O-ring or gasket. Use only 1.5ml transparent plastic tubes resistant to acetone (e.g. Eppendorf brand, polypropylene). Plastic pipettes/tips/tubes – DO NOT use plastics to handle concentrated acid stocks used for MS experiments. Plastic extracts accumulate, contaminate the stocks and, then downstream solvents, solutions, and samples.
  10. If you are unsure of your ability to prepare a sample according to these guidelines, please contact the Core Director about options for the Core to prepare your samples, or collaborate with another laboratory who are experts in proteomics sample preparation to assist with your sample preparation.

Reagents Used for Sample Preparation/Processing

Preparation/processing of protein extracts for LC/MS analysis may involve buffers, salts, enzymes, inhibitors, detergents, denaturing/chaotropic agents, reducing/alkylating/peptide reactive agents, and, sometimes, DMSO (dimethyl-sulfoxide), DMF (dimethyl-formamide), or stabilizers such as glycerol, or PEG polymers. These components (except for enzymes) are usually present at concentrations at least an order of magnitude higher than the analyzed protein/peptides and most of them are extremely detrimental to LC/MS analysis even at low concentrations; some of these agents may interfere with specific step(s) of sample processing as well. Therefore, they must be removed before LC/MS analysis at appropriate processing steps.

Necessary processing components, including antibodies (for IP) and proteolytic or other processing enzymes, should be used at sufficient, but minimal, concentrations.

Any undissolved, particular matter will clog, and potentially irreversibly damage the HPLC column and, therefore, must be removed before LC/MS analysis (e.g. by centrifugation).

Detergents (both ionic and non-ionic) severely interfere with both LC and MS parts of analysis; some of them may, as denaturing agents, interfere with the proteolytic digestion step as well. Detergents are usually difficult to remove from digested protein samples and should be used in the early steps of processing, or even avoided, where possible. Detergents can be successfully removed before proteolytic digestion of proteins using FASP columns or through protein precipitation (acetone precipitation, for example).

Salts/Buffers decrease sensitivity, greatly complicate MS analysis, and damage essential elements of MS instrument including its ion optics. Salts, buffers, other small hydrophilic components can be removed through a simple desalting process using ZipTips or equivalent before LC/MS analysis. ZipTip columns are available for resale in the PMC.

Stabilizers, e.g. glycerol, or PEG polymers, severely interfere with LC/MS analysis even at very low concentrations and are difficult to remove from prepared samples. In addition, PEG polymers tend to stick to an HPLC column and may ruin it. Usually, use of protein stabilizers is not necessary for sample processing involving proteolytic digestion and should be avoided.

DMSO, DMF interfere with MS analysis. Usually, they are not necessary for sample processing involving proteolytic digestion and should be avoided.

Protein Extracts Containing Extremely Abundant Proteins

Analysis of medium and low abundant proteins is extremely difficult/impossible in the presence of highly abundant proteins (e.g. hemoglobin in red blood cells, albumin in blood plasma); selective depletion of abundant proteins (to at least average abundance level) BEFORE proteolytic digestion of protein extracts facilitates analysis of the other proteins.

PMC STANDARDS: Quality Control (QC) Check

It is now possible to run a very small amount of your purified, undigested sample on an Agilent protein chip, which are available in the MRC. This Agilent run will not only determine the protein concentration of your samples, but will also establish if your samples are degraded or contain a particular protein at high abundance that needs to be removed. All samples to be processed in the PMC MUST be run on the Agilent Bioanalyzer in the MRC before samples will be subjected to LC/MS analysis.

Gel Bands for In-Gel Digestion
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SDS PAGE Gels

Use either pre-cast or home-made polyacrylamide gels, high-grade chemical reagents, and fresh buffers. The size of a band to be excised from a gel should not exceed these dimensions: 1mm X 1mm X 5mm. Therefore, use gels with 1 mm spacers (gel thickness) and 4-6 mm wide wells. Before use, leave any home made gels overnight on the bench at room temperature to complete polymerization step and to prevent protein crosslinking by residual radicals in the gel; during this step you must prevent the gels/wells from drying.

Coomassie Staining/Distaining

  1. Do not use ‘instant’ or ‘ready-to-use’ staining kits (“InstantBlue” or “EZBlue” reagents)
  2. Use only fresh unused colloidal Coomassie stain (GelCode Blue, Thermo) or a proteomic compatible stain, such as Sypro Ruby (Thermo or Bio-Rad)
  3. Try to stain gels for the minimum time needed until the band(s) of interest are visible
  4. De-stain bands sufficiently to clearly see the band of interest edges
  5. Do not use a microwave to speed up staining or distaining
  6. Save a high-quality image of the gel prior to band excision (optional)
  7. NEVER place gels on plastic for cutting , only cut gels on a clean glass plate

Excising the Band of Interest from a Gel

  1. Cut gels on a clean glass plate ONLY
  2. Wear gloves at all times
  3. Use a new scalpel blade and clean forceps during cutting
  4. Precisely cut out only the clearly stained band, which must not have diffused edges
  5. If band is wide, cut out its darkest, middle strip no wider than 1 mm
  6. Cut the gel band into 2-3 pieces – BUT, do not mince into small pieces
  7. Gel band pieces should not be allowed to dry. Put the excised pieces of a band into a clean 1.5 ml Eppendorf tube immediately and cap the tube tightly to keep the gel pieces moist.
  8. Gel band pieces are now ready for in-gel digestion and can be stored for 2-3 days at 4°C or six months at -20°C for further processing
Preparing Whole Cell Protein Extracts for LC/MS Analysis
*Get the downloadable instructions

 

Important Information for Sample Preparation

LC/MS analys is used for identification of proteins in analyzed samples and mapping of post-translational modifications (PTM) in identified proteins. Protein sample is digested with a proteolytic enzyme (usually trypsin) and generated peptide mix is subjected to LC/MS analysys.

Cell Lysis and Protein Extraction

The main objective of this procedure is:

  1. to efficiently lyse cells and extract proteins
  2. to preserve proteins from degradation and other uncontrolled modifications

There is no absolute single “best way” to lyse cells and extract proteins. A variety of homemade (published) and commercial buffers have been optimized for different cell (or sample) types. Conditions optimal for a specific sample should be selected.

Quality and Amount of Protein Extract Required

Processing/preparation of protein extracts for LC/MS analysis include trypsin digestion. Before trypsin digestion, protein extracts must be essentially free of a) protease inhibitors, denaturing agents, detergents, etc. that inhibit trypsin digestion, and b) protein stabilizers – glycerol, PEG, which severely interfere with MS analysis. Protein extracts can be separated from these low MW components by filtration using centrifugal filter devices of a low MWCO (e.g. FASP columns) or by acetone precipitation. Purified protein extracts are then dissolved and trypsin digested in an appropriate buffer. The required amount of digested protein in submitted samples is at least 0.2µg of cell lysate per LC/MS analysis; however, sample processing/preparation including trypsin digestion may require 5-100µg per sample (per replicate) depending on application for best result.

Protein Extracts Containing Extremely Abundant Proteins

Analysis of medium and low abundant proteins is extremely difficult (or impossible) in the presence of highly abundant proteins (e.g. hemoglobin in red blood cells, albumin in blood plasma). Selective depletion of abundant proteins from protein extracts (to at least average abundance level) is required to facilitate analysis of less abundant proteins of interest.

Preparing Whole Cell Protein Extracts – Basic Protocol

Introduction
The Thermo Scientific™ Pierce™ Mass Spec Sample Prep Kit for Cultured Cells enables reproducible processing of cultured mammalian cells for proteomic mass spectrometry (MS) analysis. The kit contains all of the necessary buffers, reagents, MS-grade enzymes; an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. 
Sample preparation can be performed in 2 alternative ways using

  • Acetone precipitation (refer to appendix A)
    or
  • FASP processing (refer to appendix B)
Appendix A- Preparing Whole Cell Protein Extracts via Acetone Precipitation

Materials Required:

  1. Pierce™ Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840
    Kit Contents (sufficient for processing 20 samples of 100μg of cell lysate protein):
  • Cell Lysis Buffer, 5ml
  • Digestion Buffer, 5ml
  • No-Weigh™ DTT, 24 microtubes, each containing 7.7mg of dithiothreitol (DTT)
  • Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA)
  • Trypsin Storage Solution, 250μl
  • Pierce Digestion Indicator, 10μg
  • Lys-C Protease, MS Grade, 20μg
  • Pierce™ Trypsin Protease, MS Grade, 2 × 20μg

Storage: Upon receipt, remove Insert A (containing Pierce Digestion Indicator, Lys-C Protease and Pierce Trypsin Protease, MS Grade) and store at -20°C. Store the remaining components at 4°C. Product is shipped on dry ice.

Additional Materials Required

  • Microcentrifuge polypropylene tubes
  • Microtip probe sonicator or nuclease (e.g., Thermo Scientific™ Pierce™ Universal Nuclease for Cell Lysis, P/N. 88700)
  • Protein assay kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)
    (Optional) Pierce Quantitative Colorimetric peptide Assay (P/N 23275)
  • Heating block
  • Chilled (-20°C) 100% acetone and 90% acetone
  • Trifluoroacetic acid (TFA)
  • Phosphate-buffered saline (PBS)
  • Vacuum concentrator (e.g., Thermo Scientific™ SpeedVac™ Vacuum Concentrator)

Procedure for Preparation of Peptides from Cultured Cells for MS Analysis

A. Material Preparation

  • Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of 100% acetone with 5mL of ultrapure water) and store at -20°C
  • Pre-chilled 100% acetone: Store 100% acetone at -20°C
  • Warm the Cell Lysis Buffer and Digestion Buffers to room temperature before use. Store buffers at 4°C. 

B. Cell Lysis

  1. Culture cells to harvest at least 100μg of protein. For best results, culture a minimum of 2 × 106 cells. Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Pellet cells using low-speed centrifugation (i.e., < 1000 × g) to prevent premature cell lysis.
  2. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100μl of Cell Lysis Buffer for a 20μl cell pellet). Pipette sample up and down to break up the cell clumps and gently vortex sample to mix.
  3. Incubate the lysate at 95°C for 5 minutes.
  4. Cool the lysate on ice for 5 minutes, spin down.
  5. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity by shearing DNA. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N 88700) to enzymatically digest DNA and RNA. If using nuclease, add 25 units of nuclease per 1ml of cell lysate and incubate at room temperature for 15 minutes.
  6. Centrifuge lysate at 16,000 × g for 10 minutes at 4°C.
  7. Carefully separate the supernatant and transfer into a new tube.
  8. Determine the protein concentration of the supernatant using established methods such as the BCA Protein Assay Kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)

C. Reduction, Alkylation and Acetone Precipitation

Note: This procedure is optimized for 100μg of cell lysate protein at 1mg/mL concentration; however, the procedure may be used for 10-200μg of cell lysate protein with an appropriate amount of reagents (DTT, IAA, Lys-C and trypsin). When using 10μg of cell lysate, a protein concentration of 0.2-1mg/ml may be used.

  1. Warm and equilibrate the Pierce Digestion Indicator to room temperature.
  2. Add 100μg of lysate protein to a polypropylene microcentrifuge tube and adjust the sample volume to 100μL using Cell Lysis Buffer to a final concentration of 1mg/ml.
  3. Add 0.5μg (0.5% w/w) of Pierce Digestion Indicator to the sample.

Note: The actual concentration is printed on the bottle label. Refer to the label to determine the required volume.

4.  Immediately before use, puncture the foil covering of the Thermo Scientific™ No-Weigh™ DTT tube with an empty pipette tip. Add 100μl of ultrapure water to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of DTT is ~500mM.

Note: To preserve DTT stability between uses, return unused micro-tubes to the pouch containing the desiccant pack.

5.  Add 2.1μl of DTT solution to the sample (final DTT concentration is ~10mM). Mix and incubate at 50°C for 45 minutes. Discard any unused DTT solution.
6.  Cool the sample to room temperature for 10 minutes, spin down.
7.  Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide tube with an empty pipette tip. Add 100μl of Cell Lysis Buffer to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of IAA is ~500mM. Protect solution from light.
8.  Add 11.5μl of IAA solution to the sample (final IAA concentration is ~50mM). Mix and incubate at room temperature for 20 minutes protected from light. Discard any unused IAA solution.
9.  After alkylation with IAA, immediately add 690μl (6 volumes) of pre-chilled (-20°C) 100% acetone to sample. Vortex tube and incubate at -20°C for four hour to overnight to precipitate proteins.
10. Centrifuge at 16,000 × g for 10 minutes at 4°C. Carefully remove acetone without dislodging the protein pellet.
11. Add 50μl of pre-chilled (-20°C) 90% acetone, vortex to mix and centrifuge at 16,000 × g for 5 minutes at 4°C.
12. Carefully remove acetone without dislodging the protein pellet. Allow the pellet to dry for 2-3 minutes and immediately proceed to Section D. Enzymatic Protein Digestion.

Note: Do not dry the acetone-precipitated protein pellet for more than 2-3 minutes; excess drying will make the pellet difficult to re-suspend in the Digestion Buffer.

D. Enzymatic Protein Digestion

1.  Add 100μl of Digestion Buffer to the acetone-precipitated protein pellet and resuspend by gentle pipetting up and down to break the pellet.

Note: An acetone-precipitated protein pellet may not completely dissolve; however, after proteolysis at 37°C, all the protein will be solubilized.

2.  Immediately before use, add 40μL of ultrapure water to the bottom of the vial containing 20μg Lys-C and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining Lys-C solution in single-use volumes at -80°C.
3. Add 2μl of Lys-C (1μg, enzyme-to-substrate ratio = 1:100) to the sample. Mix and incubate at 37°C for 2 hours.
4. Immediately before use, add 40μl of Trypsin Storage Solution to the bottom of the vial Containing 20μg trypsin and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining trypsin solution in single-use volumes at -80°C for long-term storage.
5. Add 4μl of trypsin (2μg, enzyme-to-substrate ratio = 1:50) to the sample. Mix and incubate overnight at 37°C.
6. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.
7. Speed vac the sample (106μl) for at least 2 hr. to remove the (volatile) Digestion Buffer. Record the protein amount per sample.
8. The samples are ready to be submitted to the facility for LC/MS analysis.

Appendix B- Preparing Whole Cell Protein Extracts via FASP Processing

Materials Required

  1. Pierce™ Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840 Kit Contents (sufficient for processing 20 samples of 100μg of cell lysate protein):
  • Cell Lysis Buffer, 5ml
  • Digestion Buffer, 5ml
  • No-Weigh™ DTT, 24 micro-tubes, each containing 7.7mg of dithiothreitol (DTT)
  • Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA)
  • Trypsin Storage Solution, 250μl
  • Pierce Digestion Indicator, 10μg
  • Lys-C Protease, MS Grade, 20μg
  • Pierce™ Trypsin Protease, MS Grade, 2 × 20μg

Storage: Upon receipt, remove Insert A (containing Pierce Digestion Indicator, Lys-C Protease and Pierce Trypsin Protease, MS Grade) and store at -20°C. Store the remaining components at 4°C. Product is shipped on dry ice.

2.  FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250 Kit Contents (sufficient for processing 8 samples):

  • Spin Columns, 8
  • Collection Tubes, 16
  • Tris-HCl solution,100mM, pH 8.5, 20ml
  • Ammonium Bicarbonate Solution, 50mM, 20ml – Do not use for this protocol
  • Urea, single-use, 8 micro-tubes, each containing 0.75g of urea
  • NaCl solution, 500mM, 20ml
  • Iodoacetamide (IAA), single-use, 8 micro-tubes - Do not use for this protocol

Additional Materials Required

  • Microtip probe sonicator or nuclease (e.g., Thermo Scientific™ Pierce™ Universal Nuclease for Cell Lysis, P/N. 88700)
  • Protein assay kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)
  • Pierce Quantitative Colorimetric peptide Assay (P/N 23275)
  • Micro-centrifuge polypropylene tubes
  • Heating block
  • Vortex
  • Trifluoroacetic acid (TFA)
  • Phosphate-buffered saline (PBS)
  • Vacuum concentrator (e.g., Thermo Scientific™ SpeedVac™ Vacuum Concentrator)

Procedure for Preparation of Peptides from Cultured Cells

A.  Material Preparation

  1. Warm the Cell Lysis Buffer and Digestion Buffer provided with Pierce kit to room temperature before use. Store buffers at 4°C.
  2. Urea Sample Solution: Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, also provided with the FASP Kit. Vortex the tube until all the powder dissolves.
  3. TEAB Solution, 50mM: e.g. add 1ml of 1M TEAB to 19ml of ultrapure water, mix.

B.  Cell Lysis

1. Culture cells to harvest at least 100μg of protein. For best results, culture a minimum of 2 × 106 cells.

Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Pellet cells using low-speed centrifugation (i.e., < 1000 × g) to prevent premature cell lysis.

2.  Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100μl of Cell Lysis Buffer for a 20μl cell pellet). Pipette sample up and down to break up the cell clumps and gently vortex sample to mix.
3. Incubate the lysate at 95°C for 5 minutes.
4. Cool the lysate on ice for 5 minutes, spin down.
5. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity by shearing DNA. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N 88700) to enzymatically digest DNA and RNA. If using nuclease, add 25 units of nuclease per 1ml of cell lysate and incubate at room temperature for 15 minutes.
6. Centrifuge lysate at 16,000 × g for 10 minutes at 4°C.
7. Carefully separate the supernatant and transfer into a new tube.
8. Determine the protein concentration of the supernatant using established methods such as the BCA Protein Assay Kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)

C.  Reduction and Alkylation

Note: This procedure is optimized for 100μg of cell lysate protein at 1mg/mL concentration; however, the procedure may be used for 50-100μg of cell lysate protein with an appropriate (proportional) amount of reagents (DTT, IAA, Pierce Digestion Indicator, Lys-C and trypsin).

  1. Warm and equilibrate the Pierce Digestion Indicator to room temperature.
  2. Add 100μg of lysate protein to a polypropylene microcentrifuge tube and adjust the sample volume to 100μL using Cell Lysis Buffer to a final concentration of 1mg/ml.
  3. Add 0.5μg (0.5% w/w) of Pierce Digestion Indicator to the sample (i.e. 0.005μg of Pierce Digestion Indicator per μg of sample protein).

Note: The actual concentration is printed on the bottle label. Refer to the label to determine the required volume.

4.  Immediately before use, puncture the foil covering of the Thermo Scientific™ No-Weigh™DTT tube with an empty pipette tip. Add 100μl of ultrapure water to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of DTT is ~500mM.

Note: To preserve DTT stability between uses, return unused micro-tubes to the pouch containing the desiccant pack.

5.  Add 2.1μl of 500mM DTT solution to the sample (final DTT concentration is ~10mM). Mix and incubate at 50°C for 45 minutes. Discard any unused DTT solution.
6.  Cool the sample to room temperature for 10 minutes, spin down.
7.  Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide tube with an empty pipette tip. Add 100μl of Cell Lysis Buffer to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of IAA is ~500mM. Protect solution from light.
8.  Add 11.5μl of 500mM IAA solution to the sample (final IAA concentration is ~50mM). Mix and incubate at room temperature for 20 minutes protected from light. Discard any unused IAA solution.
9.  After alkylation with IAA, immediately add 100μl of Urea Sample Solution and proceed to Section D, FASP Protein digestion

D.  FASP Protein Digestion

1.  Add 200μl of Urea Sample Solution to a Spin Filter and centrifuge at 14,000 x g for 5 min.

Note: The centrifugation times may need adjustment – keep it short but long enough to let all solvent flow through the filter to the collection tube.

2. Transfer the alkylated protein sample (step C9) into the Spin Filter. Centrifuge at 14,000 x g for 15 min. Discard the flow-through from the collection tube
3. Add 200μL of Urea Sample Solution to the Spin Filter, cap the filter, vortex and centrifuge at 14,000 x g for 12 min. Discard the flow-through from the collection tube. Repeat this step once.
4. Add 100μl of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and centrifuge at 14,000 x g for 10 min. Discard the flow-through from the collection tube. Repeat this step twice.
5.  Add 100μl of Digestion Buffer provided with Pierce kit
6. Immediately before use, add 40μL of ultrapure water to the bottom of the vial containing 20μg Lys-C and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining Lys-C solution in single-use volumes at -80°C.
7. Add 2μl of Lys-C (1μg, enzyme-to-substrate ratio = 1:100) to the sample, cap the filter, vortex 1 min, and incubate at 37°C for 2 hours.
8. Immediately before use, add 40μl of Trypsin Storage Solution to the bottom of the vial Containing 20μg trypsin and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining trypsin solution in single-use volumes at -80°C.
9. Add 4μl of trypsin (2μg, enzyme-to-substrate ratio = 1:50) to the sample, cap the filter, vortex, and Incubate overnight at 37°C. Wrap the tops of the tubes with Parafilm to minimize the effects from evaporation.
10. Transfer the Spin Filter to a new collection tube and centrifuge at 14,000 x g for 10 min. Do not discard the filtrate.
11. Add 50μl of 50 mM TEAB Solution to the Spin Filter and centrifuge at 14,000 x g for 10 min. Do not discard the combined filtrate.
12. Add 50μl 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge the Spin Filter at 14,000 x g for 10 min. Save the combined (206μl) filtrate.
13. Speed vac the sample (206μl, containing ~ 100μg of digested proteins) to ~20-50μl and desalt using C18 ZipTips (or equivalent) of appropriate capacity according to the manufacturer’s protocol.
14. Speed vac the desalted sample to dryness.
15. Resuspend the sample in 100μl of 10% acetonitrile.
16. Determine the peptide concentration in the samples using Pierce Quantitative Colorimetric Peptide Assay (P/N 23275) according to the manufacturer’s protocol.
17. Transfer at least 25μg of the digested protein sample into a new tube; record the transferred amount.
18. Speed vac the samples to dryness. The samples are ready to be submitted to the facility for LC/MS analysis.

 

Preparing Whole Cell Protein Extracts for Differential Protein Expression Analysis
*Get the downloadable instructions

 

Important Information for Sample Preparation

Differential Protein Expression Analysis determines the relative abundances of identical proteins (the molar ratios) in two or more samples representing different conditions (groups) - e.g. control vs patient, wild type vs mutant, treated vs untreated, individual time points, etc. An optimal number of biological and/or technical replicates must be analyzed per condition (group) for statistical validation of results. The investigator is expected to define the study conditions (groups) and to then determine (in collaboration with a statistician) an optimal/required number of replicates per condition. Please consult with Dr. Daniel Johnson in the Molecular Bioinformatics (mBIO) core (x8-3743) if you are unsure about statistical requirements for an experiment.

Cell Lysis and Protein Extraction

The main objective of this procedure is:

  1. to efficiently lyse cells and extract proteins
  2. to preserve proteins from degradation and other uncontrolled modifications

There is no absolute single “best way” to lyse cells and extract proteins. A variety of homemade (published) and commercial buffers have been optimized for different cell (or sample) types. Conditions optimal for a specific sample should be selected.

Quality and Amount of Protein Extract Required

Processing/preparation of protein extracts for LC/MS analysis include trypsin digestion and labeling of the generated peptides with either iTRAQ or TMT reagents. Before trypsin digestion, protein extracts must be essentially free of a) protease inhibitors, denaturing agents, detergents, etc. that inhibit trypsin digestion, b) compounds with primary amino groups and free thiols competing with peptides in labeling reaction, and c) protein stabilizers – glycerol, PEG, which severely interfere with MS analysis. Protein extracts can be separated from these low MW components by filtration using centrifugal filter devices of a low MWCO (e.g. FASP columns) or by acetone precipitation. Purified protein extracts are then dissolved and trypsin digested in an appropriate buffer. The required amount of digested protein in submitted samples is 25-100 µg per sample (per replicate).

Protein Extracts Containing Extremely Abundant Proteins

Analysis of medium and low abundant proteins is extremely difficult (or impossible) in the presence of highly abundant proteins (e.g. hemoglobin in red blood cells, albumin in blood plasma). Selective depletion of abundant proteins from protein extracts (to at least average abundance level) is required to facilitate analysis of less abundant proteins of interest.

Preparing Whole Cell Protein Extracts – Basic Protocol

Introduction
The Thermo Scientific™ Pierce™ Mass Spec Sample Prep Kit for Cultured Cells enables reproducible processing of cultured mammalian cells for proteomic mass spectrometry (MS) analysis. The kit contains all of the necessary buffers, reagents, MS-grade enzymes; an optimized protocol generates MS-compatible peptide samples from whole-cell lysates. Sample preparation can be performed in 2 alternative ways using

  • Acetone precipitation (refer to Appendix A)
    or
  • FASP processing (refer to Appendix B)

 

 

Appendix A- Preparing Whole Cell Protein Extracts via Acetone Precipitation

Materials Required

  1. Pierce™ Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840 Kit Contents (sufficient for processing 20 samples of 100μg of cell lysate protein):
  • Cell Lysis Buffer, 5ml
  • Digestion Buffer, 5ml
  • No-Weigh™ DTT, 24 microtubes, each containing 7.7mg of dithiothreitol (DTT)
  • Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA)
  • Trypsin Storage Solution, 250μl
  • Pierce Digestion Indicator, 10μg
  • Lys-C Protease, MS Grade, 20μg
  • Pierce™ Trypsin Protease, MS Grade, 2 × 20μg

Storage: Upon receipt, remove Insert A (containing Pierce Digestion Indicator, Lys-C Protease and Pierce Trypsin Protease, MS Grade) and store at -20°C. Store the remaining components at 4°C. Product is shipped on dry ice.

Additional Materials Required

Microcentrifuge polypropylene tubes
Microtip probe sonicator or nuclease (e.g., Thermo Scientific™ Pierce™ Universal Nuclease for Cell Lysis, P/N. 88700)
Protein assay kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)
Pierce Quantitative Colorimetric peptide Assay (P/N 23275)
Heating block
Chilled (-20°C) 100% acetone and 90% acetone
Trifluoroacetic acid (TFA)
Phosphate-buffered saline (PBS)
Vacuum concentrator (e.g., Thermo Scientific™ SpeedVac™ Vacuum Concentrator)

Procedure for Preparation of Peptides from Cultured Cells

A. Material Preparation

  1. Pre-chilled 90% acetone: Prepare 90% acetone in ultrapure water (e.g mix 45mL of 100% acetone with 5mL of ultrapure water) and store at -20°C.
  2. Pre-chilled 100% acetone: Store 100% acetone at -20°C.
  3. Warm the Cell Lysis Buffer and Digestion Buffers provided with Pierce kit to room temperature before use. Store buffers at 4°C.

B. Cell Lysis

  1. Culture cells to harvest at least 100μg of protein. For best results, culture a minimum of 2 × 106 cells.

Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Pellet cells using low-speed centrifugation (i.e., < 1000 × g) to prevent premature cell lysis.

2. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100μl of Cell Lysis Buffer for a 20μl cell pellet). Pipette sample up and down to break up the cell clumps and gently vortex sample to mix.
3. Incubate the lysate at 95°C for 5 minutes.
4. Cool the lysate on ice for 5 minutes, spin down.
5. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity by shearing DNA. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N 88700) to enzymatically digest DNA and RNA. If using nuclease, add 25 units of nuclease per 1ml of cell lysate and incubate at room temperature for 15 minutes.
6. Centrifuge lysate at 16,000 × g for 10 minutes at 4°C.
7. Carefully separate the supernatant and transfer into a new tube.
8. Determine the protein concentration of the supernatant using established methods such as the BCA Protein Assay Kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227).

C. Reduction, Alkylation and Acetone Precipitation

Note: This procedure is optimized for 100μg of cell lysate protein at 1mg/mL concentration; however, the procedure may be used for 50-100μg of cell lysate protein with an appropriate (proportional) amount of reagents (DTT, IAA, Pierce Digestion Indicator, Lys-C and trypsin).

  1. Warm and equilibrate the Pierce Digestion Indicator to room temperature.
  2. Add 100μg of lysate protein to a polypropylene microcentrifuge tube and adjust the sample volume to 100μL using Cell Lysis Buffer to a final concentration of 1mg/ml.
  3. Add 0.5μg (0.5% w/w) of Pierce Digestion Indicator to the sample (i.e. 0.005μg of Pierce Digestion Indicator per μg of sample protein).

Note: The actual concentration is printed on the bottle label. Refer to the label to determine the required volume.

4. Immediately before use, puncture the foil covering of the Thermo Scientific™ No-Weigh™ DTT tube with an empty pipette tip. Add 100μl of ultrapure water to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of DTT is ~500mM.

Note: To preserve DTT stability between uses, return unused micro-tubes to the pouch containing the desiccant pack.

5. Add 2.1μl of 500mM DTT solution to the sample (final DTT concentration is ~10mM). Mix and incubate at 50°C for 45 minutes. Discard any unused DTT solution.
6. Cool the sample to room temperature for 10 minutes, spin down.
7. Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide tube with an empty pipette tip. Add 100μl of Cell Lysis Buffer to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of IAA is ~500mM. Protect solution from light.
8. Add 11.5μl of 500mM IAA solution to the sample (final IAA concentration is ~50mM). Mix and incubate at room temperature for 20 minutes protected from light. Discard any unused IAA solution.
9. After alkylation with IAA, immediately add 690μl (6 volumes) of pre-chilled (-20°C) 100% acetone to sample. Vortex tube and incubate at -20°C for four hour to overnight to precipitate proteins.
10. Centrifuge at 16,000 × g for 10 minutes at 4°C. Carefully remove acetone without dislodging the protein pellet.
11. Add 50μl of pre-chilled (-20°C) 90% acetone, vortex to mix and centrifuge at 16,000 × g for 5 minutes at 4°C.
12. Carefully remove acetone without dislodging the protein pellet. Allow the pellet to dry for 2-3 minutes and immediately proceed to Section D. Enzymatic Protein Digestion.

Note: Do not dry the acetone-precipitated protein pellet for more than 2-3 minutes; excess drying will make the pellet difficult to re-suspend in the Digestion Buffer.

D. Enzymatic Protein Digestion

Add 100μl of Digestion Buffer to the acetone-precipitated protein pellet (final protein concentration is 1mg/ml). and resuspend by gentle pipetting up and down to break the pellet. It is important to dissolve as much protein as possible; water bath sonication may facilitate the process.

Note: An acetone-precipitated protein pellet may not completely dissolve; however, after proteolysis at 37°C, all the protein will be solubilized.

2. Immediately before use, add 40μL of ultrapure water to the bottom of the vial containing 20μg Lys-C and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining Lys-C solution in single-use volumes at -80°C.
3. Add 2μl of Lys-C (1μg, enzyme-to-substrate ratio = 1:100) to the sample. Mix and incubate at 37°C for 2 hours.
4. Immediately before use, add 40μl of Trypsin Storage Solution to the bottom of the vial Containing 20μg trypsin and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining trypsin solution in single-use volumes at -80°C for long-term storage.
5. Add 4μl of trypsin (2μg, enzyme-to-substrate ratio = 1:50) to the sample. Mix and incubate overnight at 37°C.
6. Acidify the sample with TFA (to 0.1%) to stop digestion, spin down.
7. Speed vac the sample (106μl) for at least 2 hr. to remove the (volatile) Digestion Buffer.
8. Resuspend the sample containing 100μg of digested proteins in 100μl of 10% acetonitrile.
9. Determine the peptide concentration in the samples using Pierce Quantitative Colorimetric Peptide Assay (P/N 23275) according to the manufacturer’s protocol.
10. Transfer at least 25μg of the digested protein sample into a new tube. Transfer equal amount of each sample into corresponding new tubes; record the transferred amount.
11. Speed vac the samples to dryness. The samples are ready to be submitted to the facility for further processing.

Appendix B- Preparing Whole Cell Protein Extracts via FASP Processing

Materials Required

  1. Pierce™ Mass Spec Sample Prep Kit for Cultured Cells, P/N 84840 Kit Contents (sufficient for processing 20 samples of 100μg of cell lysate protein):
  • Cell Lysis Buffer, 5ml
  • Digestion Buffer, 5ml
  • No-Weigh™ DTT, 24 micro-tubes, each containing 7.7mg of dithiothreitol (DTT)
  • Iodoacetamide, Single-Use, 24 microtubes, each containing 9.3mg of iodoacetamide (IAA)
  • Trypsin Storage Solution, 250μl
  • Pierce Digestion Indicator, 10μg
  • Lys-C Protease, MS Grade, 20μg
  • Pierce™ Trypsin Protease, MS Grade, 2 × 20μg

Storage: Upon receipt, remove Insert A (containing Pierce Digestion Indicator, Lys-C Protease and Pierce Trypsin Protease, MS Grade) and store at -20°C. Store the remaining components at 4°C. Product is shipped on dry ice.

2.   FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250 Kit Contents (sufficient for processing 8 samples):

  • Spin Columns, 8
  • Collection Tubes, 16
  • Tris-HCl solution,100mM, pH 8.5, 20ml
  • Urea, single-use, 8 micro-tubes, each containing 0.75g of urea
  • NaCl solution, 500mM, 20ml
  • Iodoacetamide (IAA), single-use, 8 micro-tubes - Do not use for this protocol
  • Ammonium Bicarbonate Solution, 50mM, 20ml – Do not use for this protocol

Additional Materials Required

  • Microtip probe sonicator or nuclease (e.g., Thermo Scientific™ Pierce™ Universal Nuclease for Cell Lysis, P/N. 88700)
  • Protein assay kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)
  • Pierce Quantitative Colorimetric peptide Assay (P/N 23275)
  • Micro-centrifuge polypropylene tubes
  • Heating block
  • Vortex
  • Trifluoroacetic acid (TFA)
  • Phosphate-buffered saline (PBS) Triethyl-ammonium bicarbonate (TEAB) solution, 1M (Sigma, P/N T7408-100ml)
  • Vacuum concentrator (e.g., Thermo Scientific™ SpeedVac™ Vacuum Concentrator)

Procedure for Preparation of Peptides from Cultured Cells

A. Material Preparation

  1. Warm the Cell Lysis Buffer and Digestion Buffer provided with Pierce kit to room temperature before use. Store buffers at 4°C.
  2. Urea Sample Solution: Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, also provided with the FASP Kit. Vortex the tube until all the powder dissolves.
  3. TEAB Solution, 50mM: e.g. add 1ml of 1M TEAB to 19ml of ultrapure water, mix.

B. Cell Lysis

1. Culture cells to harvest at least 100μg of protein. For best results, culture a minimum of 2 × 106 cells.

Note: Rinse cell pellets 3 times with 1X PBS to remove cell culture media. Pellet cells using low-speed centrifugation (i.e., < 1000 × g) to prevent premature cell lysis.

2. Lyse the cells by adding five cell-pellet volumes of Cell Lysis Buffer (i.e., 100μl of Cell Lysis Buffer for a 20μl cell pellet). Pipette sample up and down to break up the cell clumps and gently vortex sample to mix.
3. Incubate the lysate at 95°C for 5 minutes.
4. Cool the lysate on ice for 5 minutes, spin down..
5. Sonicate lysate on ice using a microtip probe sonicator to reduce the sample viscosity by shearing DNA. Alternatively, use Pierce Universal Nuclease for Cell Lysis (P/N 88700) to enzymatically digest DNA and RNA. If using nuclease, add 25 units of nuclease per 1ml of cell lysate and incubate at room temperature for 15 minutes.
6. Centrifuge lysate at 16,000 × g for 10 minutes at 4°C.
7. Carefully separate the supernatant and transfer into a new tube.
8. Determine the protein concentration of the supernatant using established methods such as the BCA Protein Assay Kit (e.g., Thermo Scientific™ BCA Protein Assay Kit, P/N 23227)

C. Reduction and Alkylation

Note: This procedure is optimized for 100μg of cell lysate protein at 1mg/mL concentration; however, the procedure may be used for 50-100μg of cell lysate protein with an appropriate (proportional) amount of reagents (DTT, IAA, Pierce Digestion Indicator, Lys-C and trypsin).

  1. Warm and equilibrate the Pierce Digestion Indicator to room temperature.
  2. Add 100μg of lysate protein to a polypropylene microcentrifuge tube and adjust the sample volume to 100μL using Cell Lysis Buffer to a final concentration of 1mg/ml.
  3. Add 0.5μg (0.5% w/w) of Pierce Digestion Indicator to the sample (i.e. 0.005μg of Pierce Digestion Indicator per μg of sample protein).

Note: The actual concentration is printed on the bottle label. Refer to the label to determine the required volume.

4. Immediately before use, puncture the foil covering of the Thermo Scientific™ No-Weigh™ DTT tube with an empty pipette tip. Add 100μl of ultrapure water to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of DTT is ~500mM.

Note: To preserve DTT stability between uses, return unused micro-tubes to the pouch containing the desiccant pack.

5. Add 2.1μl of 500mM DTT solution to the sample (final DTT concentration is ~10mM). Mix and incubate at 50°C for 45 minutes. Discard any unused DTT solution.
6. Cool the sample to room temperature for 10 minutes, spin down.
7. Immediately before use, puncture the foil covering of the Single-Use Iodoacetamide tube with an empty pipette tip. Add 100μl of Cell Lysis Buffer to the tube and gently pipette up and down to dissolve the contents of the tube. The final concentration of IAA is ~500mM. Protect solution from light.
8. Add 11.5μl of 500mM IAA solution to the sample (final IAA concentration is ~50mM). Mix and incubate at room temperature for 20 minutes protected from light. Discard any unused IAA solution.
9. After alkylation with IAA, immediately add 100μl of Urea Sample Solution and proceed to Section D, FASP Protein digestion

D. FASP Protein Digestion

  1. Add 200μl of Urea Sample Solution to a Spin Filter and centrifuge at 14,000 x g for 5 min.

Note: The centrifugation times may need adjustment – keep it short but long enough to let all solvent flow through the filter to the collection tube.

2. Transfer the alkylated protein sample (step C9) into the Spin Filter. Centrifuge at 14,000 x g for 15 min. Discard the flow-through from the collection tube
3. Add 200μL of Urea Sample Solution to the Spin Filter, cap the filter, vortex and centrifuge at 14,000 x g for 12 min. Discard the flow-through from the collection tube. Repeat this step once.
4. Add 100μl of 50 mM TEAB Solution to the Spin Filter, cap the filter, vortex and centrifuge at 14,000 x g for 10 min. Discard the flow-through from the collection tube. Repeat this step twice.
5. Add 100μl of Digestion Buffer provided with Pierce kit
6. Immediately before use, add 40μL of ultrapure water to the bottom of the vial containing 20μg Lys-C and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining Lys-C solution in single-use volumes at -80°C.
7. Add 2μl of Lys-C (1μg, enzyme-to-substrate ratio = 1:100) to the sample, cap the filter, vortex 1 min, and incubate at 37°C for 2 hours.
8. Immediately before use, add 40μl of Trypsin Storage Solution to the bottom of the vial Containing 20μg trypsin and incubate at room temperature for 5 minutes. Gently pipette up and down to dissolve. Store any remaining trypsin solution in single-use volumes at -80°C.
9. Add 4μl of trypsin (2μg, enzyme-to-substrate ratio = 1:50) to the sample, cap the filter, vortex, and Incubate overnight at 37°C. Wrap the tops of the tubes with Parafilm to minimize the effects from evaporation.
10. Transfer the Spin Filter to a new collection tube and centrifuge at 14,000 x g for 10 min. Do not discard the filtrate.
11. Add 50μl of 50 mM TEAB Solution to the Spin Filter and centrifuge at 14,000 x g for 10 min. Do not discard the combined filtrate.
12. Add 50μl 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge the Spin Filter at 14,000 x g for 10 min. Save the combined (206μl) filtrate.
13. Speed vac the sample (206μl, containing ~ 100μg of digested proteins) to ~20-50μl and desalt using C18 ZipTips (or equivalent) of appropriate capacity according to the manufacturer’s protocol.
14. Speed vac the desalted sample to dryness.
15. Resuspend the sample in 100μl of 10% acetonitrile.
16. Determine the peptide concentration in the samples using Pierce Quantitative Colorimetric Peptide Assay (P/N 23275) according to the manufacturer’s protocol.
17. Transfer at least 25μg of the digested protein sample into a new tube. Transfer equal amount of each sample into corresponding new tubes; record the transferred amount.
18. Speed vac the samples to dryness. The samples are ready to be submitted to the facility for further processing.

 

Protein Precipitation: Acetone Precipitation
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Introduction

Protein samples commonly contain substances that interfere with downstream applications. Several strategies exist for eliminating these substances from samples. Small soluble substances may be removed and the samples exchanged into appropriate buffers by dialysis or gel filtration (desalting columns). A variety of Thermo Scientific dialysis and desalting products are available for performing such buffer exchanges with small or large sample volumes (see Related Products). Another strategy for removing undesirable substances is to add a compound that causes protein to precipitate. After centrifugation to pellet the precipitated protein, the supernatant containing the interfering substance is removed and the protein pellet is re-dissolved in a buffer that is compatible with the downstream application. 

Several methods for protein precipitation are described in the literature. A popular method using acetone is presented here.

Important Notes:

  • Precipitation has an advantage over dialysis or desalting methods in that it enables concentration of the protein sample as well as purification from undesirable substances.
  • One disadvantage of protein precipitation is that proteins might denature, making the pellet difficult to re-solubilize.Therefore, use precipitation only for downstream applications in which solvents that aid in re-solubilizing the sample will be used (e.g., 2-D electrophoresis sample buffer, SDS-PAGE sample buffer, Pierce® BCA Protein Assay Reagent). For precipitation before performing a BCA Protein Assay, see the Tech Tip #8: Eliminate interfering substances from samples for BCA Protein Assay.
  • A single precipitation may not be sufficient to remove all types and concentrations of interfering contaminants. In such cases, repeated precipitation may be performed. However, because some sample loss will accompany each cycle of precipitation, use only the number of cycles necessary for the application.

Materials Required

  • Cold (-20°C) acetone, a volume four times that of the protein samples to be precipitated
  • Centrifuge tube, made of acetone-compatible polypropylene and able to hold five times the sample volume
  • Centrifuge and rotor for the tubes used, minimum 13,000 X g required

Protocol

  1. Cool the required volume of acetone to -20°C.
  2. Place protein sample in acetone-compatible tube.
  3. Add four times the sample volume of cold (-20°C) acetone to the tube.
  4. Vortex tube and incubate for 60 minutes to overnight at -20°C.
  5. Centrifuge 10 minutes at 13,000-15,000 X g.
  6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet.
    Optional: If additional cycles of precipitation are necessary to completely remove the interfering substance, then repeat
  7. steps 2-5 before proceeding to step 7.
  8. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it may not dissolve properly.
  9. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protein pellet.
Protein Precipitation: Methanol-Chloroform Precipitation
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Method to process 100uL of protein sample; it can be scaled up or down.

  1.  Add 400uL of Methanol to a sample of 100uL volume.

  2. Vortex well.

  3. Add 100uL of Chloroform.

  4. Vortex well.

  5. Add 300uL of ddH20. Sample should look cloudy.

  6. Vortex well.

  7. Spin 2 min. at 14,000 g

  8. Pipette off the top aqueous layer. DO NOT lose protein - protein exists between layers and may be visible as a thin wafer.

  9. Add 400uL of Methanol.

  10. Vortex well.

  11. Spin 3min. at 14,000 g

  12. Pipette as much Methanol as possible from the tube without disturbing the pellet.

  13. Speed‐Vac to dryness but avoid drying too long as this makes the pellet harder to re‐solubilize.

  14. Solubilize the pellet in buffer appropriate for downstream process.

Protein Digestion: In-Gel Trypsin Digestion
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Introduction 

In-gel digestion coupled with mass spectrometric analysis is a powerful tool for the identification and characterization of proteins.1,2 The Thermo Scientific™ In-Gel Tryptic Digestion Kit provides a complete set of reagents to perform ~150 digestions on colloidal coomassie or fluorescent dye-stained protein bands. The kit includes Thermo Scientific™ Pierce™ Trypsin Protease, MS Grade, destaining buffers, digestion buffers, reduction reagents and alkylation reagents. The methodology of this kit has been designed to function with a wide range of protein band concentrations producing complete and accurate digest for dependable mass spectrometric (MS) analysis.

Important Product Information

  • Trypsin is a serine protease that specifically cleaves peptide bonds at the carboxyl side of lysine and arginine residues. However, cleavage can be blocked or slowed by a proximal acidic, aromatic or proline residue; proline having the most significant effect. Peptide fragments with one missed cut are common and should be taken into consideration during mass analysis.
  • The Pierce Trypsin Protease, MS Grade provided in this kit displays only limited autolytic activity that should not interfere with mass spectral analysis. A trypsin fragment of mass 842.51 (m/z, M + H) will be the most common using standard conditions and can be used as an internal standard.
  • The In-Gel Tryptic Digestion Kit is designed for collodial coomassie or fluorescent dye-stained acrylamide gel slices. For protein bands stained with mass spectrometry-compatible silver stains or reversible zinc staining (Product No. 24582), alternative destaining procedures will be required.3-4
  • For SDS-PAGE separations, use polyacrylamide gels of 1mm thickness. Gels of other thicknesses may result in reduced peptide recovery yield.5
  • Reduction and alkylation of cystine residues using TCEP and IAA, respectively, improves the recovery of cystine- containing peptides from in-gel digests and minimizes the appearance of unknown masses in MS analysis from disulfide bond formation and side chain modification. Alkylation is optional, but highly recommended.6 A reliable and optimized method for reduction and alkylation, as part of the in-gel digestion protocol, is provided below. Nevertheless, alkylation can be preformed in a variety of ways dependent on the application,7-9 and no one method is optimal for all applications. Note: Alkylation with iodoacetamide increases the mass of a peptide by 57.02 for each cystine present. Acrylamide modification of cystine results in a peptide mass increase of 71.04. Note: When separating and examining proteins by 2D gel electrophoresis using alkaline conditions (i.e., pH > 8), alkylate the sample before isoelectric focusing (IEF). The use of an alternative reducing agent (e.g., hydroxyethyl disulfide) may help to avoid spurious banding in the alkaline regions caused by disulfide bond formation.7,10 Alkylation of sample before 2D electrophoresis is not required for proteins with a pI < 8.0. 

Additional Materials Required 

  • 600μL microcentrifuge tubes 
  • 50mL capped bottle or equivalent 
  • 10mL storage bottle, tube or equivalent 
  • Ultrapure water [18 megaohm (MΩ) equivalent] 

Note: Use ultrapure water in the preparation of all materials. 

Material Preparation

Note: Some of the solutions required for the In-Gel Tryptic Digestion Kit require occasional preparation while others need to be prepared just before use as needed; therefore, plan accordingly. 

Trypsin Stock: 

Pierce Trypsin Protease, MS Grade (20μg) is supplied lyophilized and may be stored in this form at -20°C for > 1 year without significant loss in activity. 

When required, prepare trypsin stock solution by hydrating the lyophilized trypsin with 20μL of the supplied Trypsin Storage Solution. This solution contains components that inactivate and protect the enzyme from autodigestion. 

To minimize freeze-thaw cycles and to increase storage stability, divide the hydrated Trypsin into four separate tubes of ~5μL each. Store each aliquot at -20°C in a nonfrost-free freezer. This solution is used to form the Trypsin Working Solution as needed (see below). 

Trypsin Working Solution:  When required, thaw a Trypsin Stock aliquot on ice. Dilute stock 10-fold by adding 45μL of ultrapure water. This solution may be stored at -20°C for 2 months without significant activity loss. 
Destaining Solution:  Mix 80mg of ammonium bicarbonate with 20mL of acetonitrile (ACN) and 20mL of ultrapure water. The Destaining Solution may be stored at 4°C for 2 months. This stock solution is sufficient for 50-100 digestions and can be prepared three times with this kit. 
Digestion Buffer:

Mix 10mg of ammonium bicarbonate with 5mL of ultrapure water (final concentration ~25mM). Digestion Buffer may be stored at 4°C for 2 months. This stock solution can be prepared three times with this kit. 

Note: An excess of Digestion Buffer is supplied to minimize the need for long-term storage and weighing minute quantities of ammonium bicarbonate. 

Reducing Buffer: 

Prepare just before use (Step B.1). Mix 3.3μL of TCEP with 30μL of Digestion Buffer for each digest to be performed. Final TCEP concentration is ~50mM. 

Note: Do not store Reducing Buffer.

Alkylation Buffer: 

Prepare just before use (Step B.3) in foil-wrapped tubes to avoid exposure to light. To avoid weighing sub-microgram quantities of IAA when a small number of samples are being processed, dissolve 7mg of IAA in 70μL water to make a 5X stock (~500mM final concentration). Dilute 7μL of the 5X stock solution with 28μL of Digestion Buffer for each digest being performed to make the final Alkylation Buffer. If greater than 10 samples are being digested simultaneously, increase the volume of stock accordingly. Excess IAA has been supplied with this kit. 

Note:Do not store the Alkylation Buffer or stock solution. 

Activated Trypsin: 

Shortly before use (Step C.3) dilute 1μL of Trypsin Working Solution with 9μL of Digestion Buffer for each sample being processed. Final concentration will be ~10ng/μL. Store Activated Trypsin on ice until use. 

Note: Do not store Activated Trypsin. 

Note: The recommended amount of trypsin used per digest is 100ng (see protocol). This amount of trypsin can be reliably used for a wide variety of protein concentration within an excised gel band. However, if protein band contains significantly less than ~20ng protein (~300fmol), 25ng of trypsin may be used per digest by diluting the Trypsin Working Solution an additional four-fold with Digestion Buffer. 

Protocol for In-gel Digest from 1D or 2D Gel Electrophoresis Separated Proteins 

A. Band Preparation and Destaining 

Note: This procedure is for collodial coomassie or fluorescent dye-stained acrylamide gel slices. Alternative destaining procedures are required for silver- or zinc-stained protein bands. See Related Thermo Scientific Products Section for a listing of compatible protein stains and the Additional Information Section for alternative destaining procedures. 

  1. Use a spot picker or scalpel to excise protein band of interest from 1D or 2D gel. Cut band into 1 X 1 to 2 X 2mm pieces. Place pieces into a 600μL receiver tube. 
    Note: Take care to include only stained region of the gel. 
  2. Add 200μL of Destaining Solution to gel pieces. Incubate sample at 37°C for 30 minutes with shaking. 
  3. Remove and discard Destaining Solution from the tube. 
  4. Repeat steps A.2-A.3. 
  5. Proceed to step B.1 or C.1. 

B. Reduction and Alkylation (Optional) 

Note: Reduction and alkylation are optional but recommended if high-sequence coverage is desired. If sample is reduced and alkylated before or during electrophoresis, it may be possible to omit these steps without affecting results. However, alkylation is inhibited or slowed by a variety of conditions, such as the presence of thiourea, SDS or a pH < 7.0; therefore, alkylation of the sample before electrophoresis may not be complete. 

  1. Prepare Reducing Buffer as described in the Material Preparation Section. Add 30μL of Reducing Buffer to the tube containing the sample and incubate at 60°C for 10 minutes. 
  2. Allow samples to cool; then remove and discard Reducing Buffer from tube. 
  3. Prepare Alkylation Buffer as described in the Material Preparation Section. Add 30μL of Alkylation Buffer to the tube. Incubate sample in the dark at room temperature for 1 hour. 
  4. Remove and discard Alkylation Buffer from tube. Wash the sample by adding 200μL Destaining Buffer to the tube. Incubate sample at 37°C for 15 minutes with shaking. 
  5. Remove and discard Destaining Buffer from tube. 
  6. Repeat Steps B.4-B.5. 
  7. Proceed to Step C.1.

C. Digestion 

  1. Shrink gel pieces by adding 50μL of acetonitrile. Incubate sample for 15 minutes at room temperature. 
  2. Carefully remove acetonitrile and allow gel pieces to air-dry for 5-10 minutes. 
  3. Prepare Activated Trypsin as described in the Material Preparation Section. Swell gel pieces by adding 10 μL of Activated Trypsin solution to the tube. Incubate sample at room temperature for 15 minutes.
    Note: If 10μL is insufficient to cover and fully swell gel pieces, increase volume accordingly. 
  4. Add 25μL Digestion Buffer to the tube. Incubate sample at 37°C for 4 hours or at 30°C overnight with shaking. 
  5. Remove digestion mixture and place in a clean tube. 
  6. (Optional) To further extract peptides, add 10μL of 1% trifluoroacetic acid or 1% formic acid solution to gel pieces and incubate for 5 minutes. Remove extraction solution and add to digestion mixture (step 5). This step also serves to inactivate trypsin, stopping additional enzymatic activity. A second extraction generally results in only a minor increase in peptide recovery. 
  7. Sample is now ready for liquid chromatographic separation and electrospray ionization mass spectrometry (LC-ESI MS) or for additional processing/clean-up as required for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or nanospray ionization mass spectrometry (see Product No. 89870).
    Note: To prevent clogging or column damage, ensure sample is free of any acrylamide pieces before applying to a LC-ESI MC system. 

Troubleshooting

Problem Cause Solution
Incomplete Digestion Insufficient enzymatic activity Increase incubation time
Ensure gel slice was dry before addition of enzyme to pull trypsin into gel slice and increase hydration volume
Enzyme was losing activity Use a new Trypsin Stock aliquot
Incorrect pH Ensure gel slice has been completely destained and Trypsin Working Solution has been diluted with digestion buffer
Residual SDS Ensure gel slice has been completely destained
Poor Mass Specturm Concentration or detection limits of application

Ensure sample is within the detection limit of the specific downstream application; concentrate digest on C18 sample prep device (Product No. 89870)

Note: Limits vary considerably based on application and instrumentation

Interfering agents Clean-up digest with C18 sample prep device 
Cited References 

1. Lahm, H.W. and Langen, H. (2000). Mass spectrometry: A tool for the identification of proteins separated by gels. Electrophoesis21:2105-14 

2. Patterson, S.D. and Aebersold, R. (2003). Proteomics: the first decade and beyond. Nat Genet33 supplement:311-23. 

3. Shevchenko, A., et al. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem68:850-8. 

4. Shevchenko, A. and Shevchenko, A. (2001). Evaluation of the efficiency of in-gel digestion of proteins by peptide isotopic labeling and MALDI mass spectrometry. Anal Biochem296:279-83. 

5. Speicher, K.D., et al. (2000). Systematic analysis of peptide recoveries from in-gel digestions for protein identifications in proteome studies. J Biomolecular Techniques.11:74-86. 

6. Sechl, S. and Chalt, B. T. (1998). Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem70:5150- 8. 

7. Herbert, B., et al. (2001). Reduction and alkylation of proteins in preparation of two-dimensional map analysis: Why, when, and how? Electrophoresis22:2046-57. 

8. Galvani, M., et al. (2001). Alkylation kinetics of proteins in preparation for two-dimensional maps: A matrix assisted desorption/ionization-time of flight-mass spectrometry investigation. Electrophoresis22:2058-65. 

9. Galvani, M., et al. (2001). Protein alkylation in the presence/absence of thiourea in proteome analysis: A matrix assisted desorption/ionization-time of flight-mass spectrometry investigation. Electrophoresis22:2066-74. 

10. Olsson, I., et al. (2002). Organic disulfides as a means to generate streak-free two-dimensional maps with narrow range basic immobilized pH gradient strips as first dimension. Proteomics2:1630-2. 

Protein Digestion: FASPTM Processing
*Get the downloadable instructions

 

Introduction

Protein Discovery’s FASP Protein Digestion Kit is for researchers who wish to solubilize whole or fractionated protein samples in SDS, digest the protein with trypsin, and analyze the resulting peptides by mass spectrometry. 

The FASP Protein Digestion Kit provides the necessary columns and buffers to carry out Universal Sample preparation as described by Wisniewski, Zoubman, Nagaraj and Mann i.The FASP Kit is compatible with a comprehensive range of biological sample types. A second protocol, included, provides instructions for digesting molecular weight fractions produed by the Gelfree® 8100 Fractionation System. 

Storage Ability

Store FASP Protein Digestion Kit materials at room temperature. Product shelf life is two years.

Protocol 1 Usage Gudielines

Proteome Extract Digestion

The following usage guidelines refer to the FASP Protein Digestion Kit when it is used in accord with the Proteome Extract Digestion protocol. 

  • The FASP Protein Digestion Kit is compatible with whole proteome extracts and other lysates from a wide variety of biological sample types. 
  • The maximum loading capacity of one FASP Protein Digestion Kit is 0.4 mg protein in up to 30 μL solution. 
  • Disulfide bonds should be reduced prior to the start of the FASP Protein Digestion protocol for best results. The FASP Protein Digestion Kit is compatible with the common reducing agents dithiothreitol, beta-mercaptoethanol, and tris(2-carboxyethyl) phosphine. If you have used Protein Discovery’s UPX™ Universal Protein Extraction Kit or YPX™ Yeast Protein Extraction Kit, then proteins have been reduced and do not require further treatment. 

Recommended Procedure 

Materials Needed 

  • FASP Protein Digestion Kit 
  • Microfuge tube 
  • Pipettor and Pipette Tips 
  • Trypsin 
  • Trifluoroacetic acid (TFA) 
  • Benchtop centrifuge capable of 14,000 x g 
  • Incubator set at 37 ˚C 
  • Vortex 

Preparing Urea Sample Solution 

Urea Sample Solution should be prepared fresh prior to digestion. 

  • Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, also provided with the FASP Kit. Vortex the tube until all the powder dissolves.  

 Preparing 10X Iodoacetamide Solution 

10X Iodoacetamide Solution should be prepared fresh prior to digestion. 

  • Make a 10X Iodoacetamide Solution by adding 100 μL Urea Sample Solution to one tube of Iodoacetamide provided with the FASP Kit. Mix and dissolve the solution by pipetting it up and down 15 times. Transfer solution to a clean, dry microfuge tube. 

Preparing Digestion Solution 

Digestion Solution should be prepared fresh prior to digestion. 

  • Make 75 μL Digestion Solution by dissolving 4 μg trypsin in 75 μL 50 mM Ammonium Bicarbonate Solution provided with the FASP Kit to a final concentration of 0.05 μg/μL. 

 Protocol 

  1. Mix up to 30 μL (0.4 mg) of a protein extract with 200 μL of 1. Urea Sample Solution in the Spin Filter and centrifuge at 14,000 x g for 15 min. 
  2. Add 200 μL of Urea Sample Solution to the Spin Filter and 2. centrifuge at 14,000 x g for 15 min. 
  3. Discard the flow-through from the collection tube3. . 
  4. Add 10 μL 10X Iodoacetamide Solution and 90 μL Urea 4. Sample Solution to the Spin Filter and vortex for 1 min; incubate without mixing for 20 min in the dark. 
  5. Centrifuge the Spin Filter at 14,000 x 5. g for 10 min. 
  6. Add 100 μL of Urea Sample Solution to the Spin Filter and 6. centrifuge at 14,000 x g for 15 min. Repeat this step twice. 
  7. Discard the flow-through from the collection tube. 7. 
  8. Add 100 μL of 50 mM Ammonium Bicarbonate Solution 8. provided with the FASP Kit to the Spin Filter and centrifuge at 14,000 x g for 10 min. Repeat this step twice. 
  9. Add 75 μL Digestion Solution (enzyme-to-protein ratio 9. 1:100) and vortex for 1 min. Wrap the tops of the tubes with Parafilm to minimize the effects from evaporation.
  10. Incubate the Spin Filter in an incubator at 37 °C for 4 – 18 h. 10.
  11. Transfer the Spin Filter to a new collection tube. 11.
  12. Add 40 μL of 50 mM Ammonium Bicarbonate Solution. 12. Centrifuge the Spin Filter at 14,000 x g for 10 min. Repeat this step once.
  13. Add 50 μL 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge the Spin Filter at 14,000 x g for 10 min.
  14. Filtrate contains digested proteins. Acidify the filtrate with 14. TFA to the desired pH and desalt.

Protocol 2 Usage Gudielines

Gelfree 8100 Fraction Digestion 

The following usage guidelines refer to the FASP Protein Digestion Kit when it is used in accord with the Gelfree 8100 Fraction Digestion protocol.

Recommended Procedure 

Materials Needed 

  • FASP Protein Digestion Kit 
  • Microfuge tube 
  • Pipettor and Pipette Tips 
  • Trypsin 
  • 0.1% Formic acid 
  • Benchtop centrifuge capable of 14,000 g 
  • Incubator set at 37 °C 
  • Vacuum concentrator 
  • Vortex 

Preparing Urea Sample Solution 

Urea Sample Solution should be prepared fresh prior to digestion. 

  • Add 1 mL Tris Hydrochloride Solution provided with the FASP Kit to one tube of Urea, also provided with the FASP Kit. Vortex the tube until all the powder dissolves. 

Preparing 10X Iodoacetamide Solution 

10X Iodoacetamide Solution should be prepared fresh prior to digestion. Make a 10X Iodoacetamide Solution by adding 100 μL Urea Sample Solution to one tube of Iodoacetamide provided with the FASP Kit. Mix and dissolve the solution by pipetting it up and down 15 times. Transfer solution to a clean, dry microfuge tube. 

Preparing Digestion Solution 

Digestion Solution should be prepared fresh prior to digestion. 

  • Make 75 μL Digestion Solution by dissolving 1 μg trypsin in 75 μL 50 mM Ammonium Bicarbonate Solution provided with the FASP Kit. 

Protocol 

  1. Add 200 μL of Urea Sample Solution to the Spin Filter and 1. centrifuge at 14,000 x g for 5 min. 
  2. Load 150 μL Gelfree fraction into the Spin Filter. Add 200 μL 2. Urea Sample Solution. Centrifuge at 14,000 x g for 25 min. 
  3. Add 200 μL of Urea Sample Solution to the Spin Filter and 3. centrifuge at 14,000 x g for 12 min. Repeat this step once.
  4. Discard the flow-through from the collection tube. 4. 
  5. Add 10 μL 10X Iodoacetamide Solution and 90 μL Urea 5. Sample Solution to the Spin Filter and vortex for 1 min; incubate without mixing for 30 min in the dark. 
  6. Centrifuge the Spin Filter at 14,000 x 6. g for 12 min. 
  7. Add 100 μL of Urea Sample Solution to the Spin Filter and 7. centrifuge at 14,000 x g for 10 min. Repeat this step twice. 
  8. Discard the flow-through from the collection tube. 8. 
  9. Add 100 μL of 50 mM Ammonium Bicarbonate Solution 9. provided with the FASP Kit to the Spin Filter and centrifuge at 14,000 x g for 10 min. Repeat this step twice. 
  10. Add 75 μL Digestion Solution (enzyme-to-protein ratio 10. 1:100) and vortex for 1 min. Wrap the tops of the tubes with Parafilm to minimize the effects from evaporation. 
  11. Incubate the Spin Filter in an incubator at 37 °C for 4 – 18 h. 11. 
  12. Transfer the Spin Filter to a new collection tube. 12. 
  13. Add 40 μL of 50 mM Ammonium Bicarbonate Solution to the 13. Spin Filter and centrifuge at 14,000 x g for 10 min. Repeat this step once. 
  14. A dd 50 μL 0.5 M Sodium Chloride Solution provided with the 14. FASP Kit and centrifuge the Spin Filter at 14,000 x g for 10 min. 
  15. Filtrate contains digested protein fraction. Use a vacuum 15. concentrator to dry the filtrate. Reconstitute sample in 20 μL of 0.1% formic acid.
Desalting Peptide Mixtures
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Introduction 

The Thermo Scientific Pierce C18 Pipette Tips enable fast and efficient capture, concentration, desalting and elution of peptides. Each tip contains a monolithic C18 reversed-phase sorbent that minimizes flow resistance and provides excellent binding and recovery characteristics at a wide range of peptide concentrations. The 100μL tip allows processing of peptide samples for desalting after digestion and before mass spectrometric analysis. The 10μL tip is ideal for off-line desalting of smaller samples. 

Matrix-assisted laser desorption ionization (MALDI-) and electrospray ionization (ESI-) mass spectrometry (MS) are vital tools for studying biological compounds because of the high sensitivity and mass accuracy. MS methods are commonly used for examining post-translational modifications and identifying proteins by peptide mapping; however, many buffers and compounds common to biological samples (e.g., urea, guanidine, NaCl, Tris, phosphate) interfere with both MALDI-MS and ESI-MS. Pierce C18 Tips remove interfering contaminants and release peptides in MS-compatible solutions, resulting in increased sensitivity and high-quality spectra. Although Pierce C18 Pipette Tips are designed primarily for MS applications, they may be used for applications such as peptide concentration and clean-up for peptide sequencing. 

Important Product Information 

  • The Pierce C18 Pipette Tips can bind up to 8μg or 80μg of total peptide in the 10μL or 100μL tip, respectively. For best results, use these tips with peptides derived from at least 20ng of protein containing at least 0.5ng of each singular peptide product. Minimum sample load requirements depend on the sensitivity limits of the downstream analysis system. Sample recovery for typical peptides is > 85%, but could be as low as 35% for hydrophilic peptides. 
  • To avoid inaccurate volumetric dispensing, do not use Pierce C18 Pipette Tips for measuring volume. 
  • For binding to C18 reversed-phase sorbents, a sample must be free of excess organic solvents such as acetonitrile (ACN) or methanol. Remove organic solvents with a centrifugal vacuum evaporator but avoid complete dryness, which might result in sample loss. Carefully dilute or resuspend sample in water with 0.1-1.0% trifluoroacetic acid (TFA) before processing with the C18 tips. 
  • For optimal results, prepare all solutions and collection tubes in advance and proceed with the entire procedure in a timely manner. Do not introduce air through the membrane during any portion of the procedure for optimum flow and peptide recovery. 
  • Plastics used during handling of peptide samples can introduce contaminants that interfere with MS analysis and result in sample loss from nonspecific adsorption. Use high-quality receiver tubes. If necessary, receiver tubes used for the final collection may be rinsed with 70% ACN/0.1% formic acid before use. Minimizing sample transfers and freeze-thaw cycles before analysis will help minimize plastic contamination and sample loss. 

Additional Materials Required 

  • 10μL or 100μL pipettor 
  • Ultrapure water 
  • Acetonitrile (ACN) 
  • Trifluoroacetic acid (TFA) 
  • Autosampler vials or 0.5mL, 1.5mL microcentrifuge tubes 
  • Formic acid or acetic acid 
  • MALDI matrix (optional) 
  • Methanol (optional) 

Material Preparation 

  • Sample treatment solution: 2.5% TFA 
  • Wetting solution: 50:50 ACN:water; 20μL or 200μL per sample 
  • Equilibration solution: 0.1% TFA in water; 20μL or 200μL per sample 
  • Rinse solution: 0.1% TFA in 5% ACN:water; 20μL or 200μL per sample 
  • Elution solution: 0.1% TFA in 50-95% ACN:water for MALDI-MS or 0.1% FA in 50-75% ACN:water for ESI-MS, up to 100μL per sample 

Procedure for the 10μL C18 Tips 

Note: For optimal flow and peptide recovery, do not introduce air through the membrane at any time during the procedure. 

  1. Set the pipettor to 10μL and secure the pipette tip tightly to the end of the pipettor for optimum tip-to-pipettor seal and sample aspiration. 
  2. Adjust sample to 0.1-1.0% TFA using 2.5% TFA. 
  3. Wet tip by aspirating 10μL of 50% ACN in water and then discarding solvent. Repeat once. 
  4. Equilibrate tip by aspirating 10μL of 0.1% TFA and discarding solvent. Repeat once. 
  5. Aspirate up to 10μL of sample (prepared in Step 2) into the C18 tip. For maximum efficiency, dispense and aspirate sample for 3-10 cycles. 
  6. Rinse the tip by aspirating 10μL of 0.1% TFA/5% ACN and discarding solvent. Repeat once. 
  7. Elute the sample as follows: 
    MALDI-TOF analysis: Slowly aspirate 2-10μL of 0.1% TFA in 50-95% ACN elution solution with or without matrix and dispense directly onto a MALDI plate. 
    LC/MS or LC/MS/MS analysis: Slowly aspirate 2-10μL of 0.1% formic acid or 0.1% acetic acid in a 50-95% ACN or methanol and dispense into an autosampler vial or well plate.

Procedure for the 100μL C18 Tips 

Note: For optimal flow and peptide recovery, do not introduce air through the membrane at any time during the procedure. 

  1. Set the pipettor to 100μL and secure the pipette tip tightly to the end of the pipettor for optimum tip-to-pipettor seal and sample aspiration. 
  2. Adjust sample to 0.1-1.0% TFA using 2.5% TFA. 
  3. Wet tip by aspirating 100μL of 50% ACN in water and then discarding solvent. Repeat once. 
  4. Equilibrate tip by aspirating 100μL of 0.1% TFA and discarding solvent. Repeat once. 
  5. Aspirate up to 100μL of sample (prepared in Step 2) into the C18 tip. For maximum efficiency, dispense and aspirate sample for 3-10 cycles. 
  6. Rinse the tip by aspirating 100μL of 0.1% TFA/5% ACN and discarding solvent. Repeat once. 
  7. Elute the sample as follows: 
    MALDI-TOF analysis: Slowly aspirate 5-100μL of 0.1% TFA in 50-95% ACN elution solution with or without matrix and dispense directly onto a MALDI plate. 
    LC/MS or LC/MS/MS analysis: Slowly aspirate 5-100μL of 0.1% formic acid or 0.1% acetic acid in a 50-95% ACN or methanol and dispense into an autosampler vial or well plate. 

Troubleshooting

Problem Possible Cause Solution
Poor or incomplete sample binding High pH, lack of ion-pairing agents Ensure TFA was added to sample 
Tip not sufficiently wetted Check buffers and prepare new tip
Sample contains organic solvent Dry sample and resuspend in 10μL or 100μL of 0.1-1.0% TFA
Sorbent became dry before adding sample Ensure that air is not drawn into the tip and that sorbent does not dry during sample processing
Sample not sufficiently hydrophobic to bind C18 sorbent None 
Poor or incomplete sample recovery Highly hydrophobic sample Use 70% ACN for elution
Peptides binding to plastics can cause significant loss at low peptide concentrations Minimize contact with plastics, excessive drying and storage at low concentrations (i.e., < 300fmol)
Detection limits of the specific application Ensure sample is within the detection limit of the specific downstream application − limits vary considerably based on application and instrumentation
High pH RP Fractionation of Peptide Mixtures
*Get the downloadable instructions

 

Introduction 

The Thermo Scientific™ Pierce™ High pH Reversed-Phase Peptide Fractionation Kit provides an optimized fractionation protocol and reagents to increase the number of proteins identified from complex samples by liquid chromatography-mass spectrometry (LC-MS) analysis. High-pH reversed-phase chromatography is a robust method of peptide fractionation that separates peptides by hydrophobicity and provides excellent orthogonality to low-pH reversed-phase LC-MS gradients. In contrast to strong cation exchange (SCX) fractionation, high-pH reversed-phase fractions do not require an additional desalting step before LC-MS analysis. 

The kit includes a high-pH solution (0.1% triethylamine) and 12 spin columns containing pH-resistant, reversed-phase resin. Each reversed-phase fractionation spin column enables fractionation of 10-100μg of peptide sample using a microcentrifuge. Native, phosphorylated, Thermo Scientific™ Tandem Mass Tag™ (TMT™)-labeled, and other complex peptide mixture samples can be fractionated using the kit. Combining the search results generated by the individual fractions improves protein sequence coverage and increases the number of identified proteins relative to unfractionated samples. 

Procedure Summary 

Proteolytic digests of proteins extracted from cells or tissues are loaded onto an equilibrated, high-pH, reversed-phase fractionation spin column. Peptides are bound to the hydrophobic resin under aqueous conditions and desalted by washing the column with water by low-speed centrifugation. A step gradient of increasing acetonitrile concentrations in a volatile high-pH elution solution is then applied to the columns to elute bound peptides into eight different fractions collected by centrifugation. Each fraction is then dried in a vacuum centrifuge (e.g., Thermo Scientific™ SpeedVac™ Vacuum Concentrator) and stored until analysis by mass spectrometry. During LC-MS analysis, peptides in each high-pH fraction are further separated using a low-pH gradient, thus reducing the overall sample complexity and improving the ability to identify low-abundant peptides.

Important Product Information 

  • Do not exceed the recommended centrifugation speeds because this may damage the column frit, causing the resin material to leak, leading to sample loss and/or damage to the LC system. 
  • Use low protein-binding microcentrifuge tubes to ensure maximum sample recovery. 
  • Store high-pH buffers in polypropylene tubes at room temperature. Do not store high-pH buffers in glass vessels. 
  • Avoid sample contamination and direct skin contact with solvents and chemicals. Always wear gloves when handling the spin columns and samples. 

Additional Materials Required 

  • Trifluoroacetic acid (Product No. 28904) 
  • Acetonitrile (ACN), LC-MS Grade (Product No. 51101) 
  • Water, LC-MS Grade (Product No. 51140) 
  • Thermo Scientific™ Pierce™ Low Protein Binding Microcentrifuge Tubes, 2.0mL (Product No. 88379 or 88380) 
  • Microcentrifuge with adjustable rotor speed up to 7,000 X
  • Vacuum centrifuge 

Material Preparation 

Trifluoroacetic acid (TFA), 0.1%

Prepare 10mL of equilibration solution by adding 10μL of TFA to 10mL of water. Volume is sufficient for equilibration of 12 columns.

 Table 1. Preparation of elution solutions for unlabeled, native peptides. 

Fraction No. Acetonitrile (%) Acetonitrile (μL) Triethylamine (0.1%) (μL)
1 5.0 50 950
2 7.5 75 925
3 10.0 100 900
4 12.5 125 875
5 15.0 150 850
6 17.5 175 825
7 20.0 200 800
8 50.0 500 500

Table 2. Preparation of elution solutions for Thermo Scientific TMT-labeled peptides.

Fraction No. Acetonitrile (%) Acetonitrile (μL) Triethylamine (0.1%) (μL)
Wash 5.0 50 950
1 10.0 100 900
2 12.5 125 875
3 15.0 150 850
4 17.5 175 825
5 20.0 200 800
6 22.5 225 775
7 25.0 250 750
8 50.0 500 500

Fractionation of Proteolytic Digests 

  1. Conditioning of the Spin Columns 
    Note: Do not exceed recommended centrifugation speeds.
  1. Remove the protective white tip from the bottom of the column and discard. Place the column into a 2.0mL sample tube. 
  2. Centrifuge at 5000 X g for 2 minutes to remove the solution and pack the resin material. Discard the liquid. 
  3. Remove the top screw cap and load 300μL of ACN into the column. Replace the cap, place the spin column back into a 2.0mL sample tube and centrifuge at 5000 X g for 2 minutes. Discard ACN and repeat wash step.
  4. Wash the spin column twice with 0.1% TFA solution, as described in Step 3. The column is now conditioned and ready for use.

B.Fractionation of Digest Samples
Note: Each sample requires 300μL of each elution solution. If more than three samples require fractionation, prepare larger volumes of the elution solutions to accommodate all samples.

  1. Prepare elution solutions according to Table 1 or Table 2 depending on sample type.
  2. Dissolve 10-100μg of digested sample in 300μL of 0.1% TFA solution.
    Note: Peptide samples need to be completely dissolved and free of organic solvent (e.g.,ACN, DSMO, etc.). If thesample contains urea, make sure that the final concentrationof urea is<1M.
  3. Place the spincolumn into a new 2.0mL sample tube. Load 300μL of the sample solutiononto the column, replace the top cap and centrifuge at 3000 X g for 2 minutes. Retain eluate as “flow-through” fraction.
  4. Place the column into a new 2.0mL sample tube. Load 300μL ofwater onto the columnand centrifugeagain to collect the wash. Retain eluate as “wash” fraction.
    Note: TMT-labeled samples require an additional column washwith 300μL of 5%ACN, 0.1%TEA (see Table 2) toremove unreacted TMT reagent.
  5. Place the column into a new 2.0mLsample tube. Load 300μL of the appropriate elution solution (e.g.,5% ACN,0.1%TEA) and centrifuge at 3000 X for 2 minutes to collect the fraction.
  6. Repeat Step 5 for the remaining step gradient fractions using the appropriate elution solutionsfrom Table 1or Table 2 in new 2.0mL sample tubes.
  7. Evaporate the liquid contents of each sample tube to dryness using vacuum centrifugation (e.g., Speed Vacconcentrator).
  8. Re-suspend dry samples in an appropriate volume of 0.1% formic acid (FA) before LC-MS analysis.
  9. Optional: Determine the peptide concentration and yield with a peptide quantitation assay, so equivalent sample amounts can be analyzed by LC-MS.

Troubleshooting

Problem Possible Cause Solution
Low peptide yields Low protein yield following lysis and protein extraction procedure Estimate protein concentration using BCA assay
Lyophilized/dried peptide samples were not completely solubilized before sample loading onto the spin column Increase vortexing/sonication time to completely dissolve the dried peptide sample
Unsuccessful fractionation Incorrect centrifuge speeds used for fractionation

Ensure proper centrifuge speed is used [in (× g)]. To convert from revolutions per minute (rpm) to g, use the following formula: 

g = (1.118 × 10-5) RS

where g is the relative centrifugal force, R is the rotor radius in centimeters, and S is the centrifuge speed in rpm. For example, centrifugation of a sample at 5,000 rpm in a microcentrifuge having a rotor radius of 7cm will deliver a centrifugal force of 1,957 × g

Low peptide/protein identification numbers


Low sample load (< 10μg) Estimate peptide concentration using the Thermo Scientific™ Pierce™ Quantitative Fluorometric Peptide Assay (Product No. 23290) or Thermo Scientific™ Pierce™ Quantitative Colorimetric Peptide Assay (Product No. 23275)
Use low protein-binding tubes for handling of the samples and fraction collection 
Incorrect chromatography or mass spectrometer instrument settings Consult instrument user manuals or online resources to determine the optimal instrument settings for your system
Verify LC-MS system performance with the Thermo Scientific™ Pierce™ HeLa Digest Protein Standard (Product No. 88328)

 

Last Published: Oct 31, 2017