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. 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 and submit it to the Proteus LIMS system.
Reagents needed for gel spot digestion:
** indicates reagents only needed if reducing/alkylating **
|| indicates reagents which improve digestion, elution, and/or mass spec response, but are not absolutely necessary ||
- 50% acetonitrile (AcN), plus [0.1% trifluoroacetic acid (TFA) for samples that will be analyzed by MALDI TOF-TOF (4800, 5800); OR 0.1% formic acid for samples that will be analyzed by ESI (electrospray, nanospray, i.e., on the QTrap or on the Waters Synapt)
- Appropriate Gel Stain (see right column 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 or Formic acid ||
**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 (Download first protocol 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.
- Download Summary Procedure if you are reducing/alkylating your samples before electrophoresis >>
- Download Summary Procedure if you are reducing/alkylating your samples after electrophoresis >>
RESULTING PEAK MWs from the Mass Spectrum, and/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 various publically available Protein Mass Spec Search Engines. In house, we search almost exclusively using the Paragon Algorithm contained in Protein Pilot software package; however, there are other search engines available if you wish to preform additional searches of your own data on your own:
- MS-FIT programs at the UCSF Protein Prospector site
- MASCOT at Matrix Sciences
- Peptide Search at Heidelberg
- Profound at the Prowl site at Rockefeller
- PeptIdent or MultiIdent at the Expasy site
- MassSearch at the CBRG in Switzerland (Computational Biology Research Group), or
- MOWSE at the UK Human Genome Mapping Project Resource Centre