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Core Research Facilities

Proteins & Mass Spectrometry

Mass spectrometry and tandem MS/MS

In-Gel Digestion Protocols

General Sample preparation requirements:

  • Small amounts of buffers (e.g. <50mM phosphate,Tris, NaCl) can be tolerated in MALDI-TOF samples, MUCH less in samples for Electrospray ionization (ESI or Nanospray).
    Avoid detergents and glycerol. If detergents must be used, octyl glucoside is the best choice for compatibility with MALDI-TOF-MS.

  • Sample concentrations should be 1-10µM but only a few µl are needed - the sensitivity of the techniques is usually such that peaks at 0.1-10 pmols/µl in a simple mix can be detected easily, with sensitivities possible down to high attomole amounts of material with special preparation.

  • However, peak suppression effects from buffer components or other peaks, as well as differences in the chemical nature of each peptide sequence, means that not all peaks in a complex mix will necessarily be detected.

Detailed sample-preparation & digestion protocols, citations

In-gel Digestion protocol

Lack of careful sample preparation is THE biggest barrier to successful protein identification, particularly contamination of samples with keratins from skin and hair (work in a hood if possible, wear gloves and cover your head), contamination from bacterial/mold growth in old solutions (make fresh solutions up, including gel destaining solutions, etc.), and exposure to detergents (do not re-use laboratory containers for sample preparation). Mass spectroscopy can detect low femtomole or even attomole amounts of material, and the more contaminant peaks you have, the less likely it is that the existing algorithms can correctly identify your protein of interest from the surrounding noise. Although the particular contaminants seen can vary greatly, a list of common contaminant peaks can be found at http://www.proteinworks.com/contamin.htm; more complete lists are available in the MALDI Methods notebook in our facility.

A number of similar protocols with different details have been successfully used at various Mass Spec facilities (click here to see other protocol examples in a new window), but the procedure below has been used successfully here in our facility at the Penn State College of Medicine to identify unknown proteins from gels, and represents a synthesis of multiple published methods and experimental determinations of optimal conditions (see in particular "Systematic Analysis of Peptide Recoveries from In-Gel Digestions for Protein Identifications in Proteome Studies" Kaye D. Speicher, Olivera Kolbas, Sandra Harper, and David W. Speicher at http://www.abrf.org/JBT/2000/june00/jun00speicher.html, but note that we have subsequently found that for optimal sample cleanup with ZipTip SCX tips, complete evaporation and 3X resuspension in 200 µl H2O to get rid of NH4CO3 gives much better overall MS peak spectra, even though the Speicher paper shows that complete evaporation gives lower radioactive peptide yields compared to partial evaporation).

It can be used for either bands from 1D gels (which are almost certain to have multiple proteins contained in it, complicating subsequent analysis, although we have successful identified multiple proteins from single1D gel bands), or spots from 2D gels (preferred, since it cuts down (but doesn’t eliminate) the problem of multiple co-electrophoresing proteins, and provides additional information about pI which is significant in subsequent database searching).

Note that you must provide material for MALDI-TOF analysis from both a sample band/spot and an identically-treated negative control band/spot (no protein, or unrelated protein band/spot) to help eliminate potential artifact peaks. It is also recommended (but not required) that you provide an identically-treated positive control such as BSA or another known protein which stains to approximately the same level as your unknown protein of interest. YOU MUST FILL OUT A SAMPLE SUBMISSION FORM!

Reagents needed for gel spot digestion:
 ** indicates reagents only needed if reducing/alkylating **
|| indicates regents which improve digestion, elution, and/or mass spec response, but not absolutely necessary ||
 50% acetonitrile (AcN), 0.1% trifluoroacetic acid (TFA)
 Appropriate Gel Stain (see blue box 3 below)
 200 mM ammonium bicarbonate (NH4HCO3) pH 8 mixed 1:1 with Acetonitrile (AcN) to give
                  final concentration of 100 mM  ammonium bicarbonate in 50% AcN
600 µL of 25 mM ammonium bicarbonate, pH 8.0
20-50 ul of 0.02 µg/µl of Promega Sequencing grade modified trypsin in 10% AcN, 40 mM NH4HCO3 pH 8;
                   plus ||0.1% w/v n-octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside), 0.1% TFA ||
 **2 mM TCEP (Tris(2-carboxyethyl)phosphine, Sigma #C4706) in 25 mM ammonium bicarbonate (pH 8.0)
                  (Alternate reagent - use 10 mM DTT, 25 mM ammonium bicarbonate (pH 8.0))**
**100 µL of 20 mM iodoacetamide in 25 mM ammonium bicarbonate (pH 8.0)**

NOTE: While highly successful results have been obtained on a routine basis for either of the procedures below, we highly recommend reduction/alkylation before electrophoresis (yellow column below). This is normally done with most 2D gel methods before the first dimension anyway, but even with 1D gels, doing the reduction/alklyation steps before putting samples onto your gel will reduce the amount of open-tube sample manipulation of gel spots after the spots are cut out, and therefore reduce the likelihood of keratin contamination of your samples.
 

  Summary Procedure if you are reducing/alkylating your samples before electrophoresis (this is typically done before the first dimension on 2D gels, for example)   Summary Procedure if you are reducing/alkylating your samples after electrophoresis (The reduction/alkylation steps below can be skipped, as it is not always necessary to perform in order to ID a protein from a gel spot; however, the reduction/alkylation will tend to increase the efficiency of trypsin digestion somewhat and will sometimes contribute to the identification of additional peptides from the protein spot).
1). Prepare gel ~ 1 day ahead of time to allow maximal polymerization. 1). Prepare gel ~ 1 day ahead of time to allow maximal polymerization.
2). Wash clear microfuge tubes with 50% acetonitrile (AcN), 0.1% trifluoroacetic acid (TFA) 2). Wash clear microfuge tubes with 50% acetonitrile (AcN), 0.1% trifluoroacetic acid (TFA)
3). Reduce sample in reducing buffer (final concentration 25 mM Tris, 0.5% SDS, 1 mM tris[2-carboxyethyl] phosphine hydrochloride [TCEP-HCl], pH 8.0) for ~10 minutes at 37°C. (N.B., we have not yet tried this, but Pierce now sells agarose-linked TCEP (Pierce # 77712). If you use this, then you can remove the TCEP before step 4, which will increase the efficiency of alkylation) 3). Load samples and run gel, then stain (Epicocconone-based stains (LavaPurple or Deep Purple), Sypro Ruby, Krypton, Colloidal Coomassie Blue (G250),  Ruthenium II,  or non-formaldehyde Silver Stain (using carbohydrazide instead of formaldehyde for reduction/development) give overall best results (see "About the mechanism of interference of silver staining with peptide mass spectrometry"Sophie Richert 1 ,Sylvie Luche 2 ,Mireille Chevallet 2 ,Alain Van Dorsselaer 1 , Emmanuelle Leize-Wagner 1 and Thierry Rabilloud 2Proteomics 2004,4,909 –916 909 for details) followed by  Coomassie R250,  Sypro Red, Sypro Orange, Silver Stain Plus, negative Zinc or copper staining at lesser sensitivity or increased interference with subsequent MALDI-TOF. A recent comparison suggests that Deep Purple may perform better that SyproRuby, at least with subsequent LC/MS/MS detection (Rapid Commun. Mass Spectrom. 22: 881–886, 2008).

 DO NOT USE GLUTARALDEHYDE OR FORMALDEHYDE-CONTAINING SILVER STAINS!). Silver stains with low formaldehyde content, e.g., "Silver Stain Plus", can be used but they are suboptimal and give weaker MS signals than gel spots coming from the preferred stains above; however, we have ID'd thousands of proteins from such Silver Stain Plus stained spots.
4). Alkylate sample by adding pH 8.0 iodoacetamide to 5 mM (final concentration). If your eventual gel loading volume will tolerate it, add this in such a way as to reduce the previous TCEP concentration to 0.1-0.2 mM. Incubate in the dark for ~15 minutes @ 37°, then add 5X gel loading buffer and incubate an additional ~15 minutes @ 37° in the dark. 4). Cut out bands/spots of interest at the margin of detectable stain. Measure/approximate gel slice volumes. Put in pre-washed 500 µl microfuge tube.
5). Load samples and run gel, then stain (Epicocconone-based stains (LavaPurple or Deep Purple), Sypro Ruby, Krypton, Colloidal Coomassie Blue (G250),  Ruthenium II,  or non-formaldehyde Silver Stain (using carbohydrazide instead of formaldehyde for reduction/development) give overall best results (see "About the mechanism of interference of silver staining with peptide mass spectrometry"Sophie Richert 1 ,Sylvie Luche 2 ,Mireille Chevallet 2 ,Alain Van Dorsselaer 1 , Emmanuelle Leize-Wagner 1 and Thierry Rabilloud 2Proteomics 2004,4,909 –916 909 for details) followed by  Coomassie R250,  Sypro Red, Sypro Orange, Silver Stain Plus, negative Zinc or copper staining at lesser sensitivity or increased interference with subsequent MALDI-TOF. A recent comparison suggests that Deep Purple may perform better that SyproRuby, at least with subsequent LC/MS/MS detection (Rapid Commun. Mass Spectrom. 22: 881–886, 2008).

 DO NOT USE GLUTARALDEHYDE OR FORMALDEHYDE-CONTAINING SILVER STAINS!). Silver stains with low formaldehyde content, e.g., "Silver Stain Plus", can be used but they are suboptimal and give weaker MS signals than gel spots coming from the preferred stains above; however, we have ID'd thousands of proteins from such Silver Stain Plus stained spots.

 		 5). Destain two times with 200 µl of {200 mM ammonium bicarbonate (NH4HCO3) pH 8 mixed 1:1 with Acetonitrile (AcN) to give final concentration in 200µl of 100 mM  ammonium bicarbonate in 50% AcN} 45 min @ 37° C.

After 2nd destain, remove liquid, then dry gel slice completely in SpeedVac.
6). Cut out bands/spots of interest at the margin of detectable stain, measure/approximate gel slice volume, and put in pre-washed 500 µl microfuge tube 6). Reduce: Add 100 µL of 2 mM TCEP (Tris(2-carboxyethyl)phosphine, Sigma #C4706) in 25 mM ammonium bicarbonate (pH 8.0) to the dried gel and incubate 15 minutes at 37°C with agitation; remove supernatant. (Alternate procedure - use 10 mM DTT, 25 mM ammonium bicarbonate (pH 8.0) for 15 minutes @ 37°C.)
7). Destain two times with 200 µl of {200 mM ammonium bicarbonate (NH4HCO3) pH 8 mixed 1:1 with Acetonitrile (AcN) to give final concentration in 200µl of 100 mM  ammonium bicarbonate in 50% AcN} 45 min @ 37° C.

After 2nd destain, remove liquid, then dry gel slice completely in SpeedVac.		 7). Alkylate: add 100 µL of 20 mM iodoacetamide in 25 mM ammonium bicarbonate (pH 8.0) and incubate for 30 minutes at 37°C in the dark; discard the supernatant; Wash gel band three times with 200 µL of 25 mM ammonium bicarbonate for 15 minutes, each with agitation; then dry gel slice completely in SpeedVac.
8). Rehydrate gel slice in 20 µl or 1.5 X original gel slice volume (whichever is greater) of 0.02 µg/µl of Promega Sequencing grade modified trypsin in 10% AcN, 40 mM NH4HCO3 pH 8; 0.1% w/v n-octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside) for 1 h at room temperature or on ice to allow the concentrated trypsin to diffuse into the gel slice. 8). Rehydrate gel slice in 20 µl or 1.5 X original gel slice volume (whichever is greater) of 0.02 µg/µl of Promega Sequencing grade modified trypsin in 10% AcN, 40 mM NH4HCO3 pH 8; 0.1% w/v n-octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside) for 1 h at room temperature or on ice to allow the concentrated trypsin to diffuse into the gel slice.
9). Remove any trypsin-containing liquid that hasn't been absorbed into the gel slice, then cover the gel slice with 50 µl 10% AcN, 40 mM NH4HCO3 pH 8; (for optimal results, include 0.1% w/v n-octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside) in the 50µl as well), and incubate with agitation at least 3 hours @ 48° C - longer is okay if more convenient (Alternately, incubate 16-18 h @ 37°C - although note that, for any given time of incubation, trypsin efficiency is higher at 48°C, see e.g. Kinetic characterization of sequencing grade modified trypsin. Finehout EJ, Cantor JR, Lee KH.Proteomics. 2005 Jun;5(9):2319-21.) 9). Remove any trypsin-containing liquid that hasn't been absorbed into the gel slice,  then cover the gel slice with 50 µl 10% AcN, 40 mM NH4HCO3 pH 8; (for optimal results, include 0.1% w/v n-octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside) in the 50µl as well), and incubate with agitation at least 3 hours @ 48° C  - longer is okay if more convenient (Alternately, incubate 16-18 h @ 37°C - although note that, for any given time of incubation, trypsin efficiency is higher at 48°C, see e.g. Kinetic characterization of sequencing grade modified trypsin. Finehout EJ, Cantor JR, Lee KH.Proteomics. 2005 Jun;5(9):2319-21.)
10). Remove the supernatant and put in fresh pre-washed 0.5 ml tube (extract 1) 10). Remove the supernatant and put in fresh pre-washed 0.5 ml tube (extract 1)
11). (optional) Add 50µl 0.1% TFA to gel slice, incubate 1 h @ 37°C. remove supernatant (extract 2) and combine with extract 1 in 0.5 ml tube. (this may extract ~15% more digested material from your sample). 11). (optional) Add 50µl 0.1% TFA to gel slice, incubate 1 h @ 37°C. remove supernatant (extract 2) and combine with extract 1 in 0.5 ml tube. (this may extract ~15% more digested material from your sample).
12). SpeedVac to dryness. Resuspend in ~200 µl H2O, then SpeedVac to dryness again. Repeat this 3X, and Speedvac the final resuspension to ~10 µl - we mark 10 µl on the 0.5 ml microfuge tube used to estimate 10 µl. (This procedure removes the NH4HCO3 (and AcN), which we have found can interfere with subsequent binding to the strong cation exchange ZipTip SCX tips we use for sample cleanup.) NOTE THAT THIS IS A CHANGE FROM PREVIOUSLY RECOMMENDED "Dry only to 25% original volume" INSTRUCTIONS from the Speicher article cited above. 12). SpeedVac the supernatant(s) to dryness (NOT the gel slice. Resuspend in ~200 µl H2O, then SpeedVac to dryness again. Repeat this 3X, and Speedvac the final resuspension to ~10 µl - we mark 10 µl on the 0.5 ml microfuge tube used to estimate 10 µl. (This procedure removes the NH4HCO3 (and AcN), which we have found can interfere with subsequent binding to the strong cation exchange ZipTip SCX tips we use for sample cleanup.) NOTE THAT THIS IS A CHANGE FROM PREVIOUSLY RECOMMENDED "Dry only to 25% original volume" INSTRUCTIONS  from the Speicher article cited above.
13). ZipTip concentrate and clean digest (normally, we do this in the facility, so you can just give Step 12 above to us) 13). ZipTip SCX concentrate and clean digest (normally, we do this in the facility, so you can just give Step 12 above to us)
  ADVANTAGES: Pre-alkylation prevents (slow) alkylation by exposure to acrylamide and cross-linkers, thus preventing a mixed mass population for specific fragments (some modified by acrylamide +71, some by iodoacetamide +57).   ADVANTAGES: Since the protein sample is fixed in the gel slice, the TCEP can be removed before adding iodoacetamide, thus insuring optimal reduction AND subsequent alkylation.
  DISADVANTAGE: Since the protein sample is not fixed in a gel slice, the TCEP cannot be removed before adding iodoacetamide, and TCEP (or DTT) over ~0.1 mM can inhibit subsequent alkylation by iodoacetamide. [N.B., we have not yet tried this, but the agarose-linked TCEP (Pierce # 77712) would theoretically overcome this.]   DISADVANTAGE: Exposure to unpolymerized acrylamide/bis-acrylamide (some of which always remains even in polymerized gels) causes slow alkylation of cysteines (+71 adduct), which thus produces a mixed population of Cys- and modified-Cys containing fragments and decreases the power of subsequent mass spec analysis (both decreased peak sizes and a more complex mix to deconvolute).
  NOTES: Other reducing agents (1 mM tributylphosphine, 10 mM DTT) have been used successfully elsewhere, and other alkylating agents can be substituted if +57 addition may confound subsequent analysis of other adducts (e.g., iodoacetic acid, vinylpyridine, or even more extensive exposure to unpolymerized acylamide.) Successful Mass Spec determinations have also been done without any alkylation, and without adding AcN or n-ocylglucoside to the trypsin digest mix - the above simply represents a "best" protocol that is theoretically optimized and has worked well for us.   NOTES: Other reducing agents (1 mM tributylphosphine, 10 mM DTT) have been used successfully elsewhere, and other alkylating agents can be substituted if +57 addition may confound subsequent analysis of other adducts (e.g., iodoacetic acid, vinylpyridine, or even more extensive exposure to unpolymerized acylamide.) Successful Mass Spec determinations have also been done without any alkylation, and without adding AcN or n-ocylglucoside to the trypsin digest mix - the above simply represents a "best" protocol that is theoretically optimized and has worked well for us.

RESULTING PEAK MWs from the Mass Spectrum, or masses from ms/ms fragment spectra (usually using ms/ms fragmentation of the top 5-10 peaks at most) can then be used to search for corresponding proteins using either

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