Biopolymers & Proteomics Laboratory

The David H.Koch Institute for Integrative Cancer Research at MIT

Bldg 76 Room 181

Telephone: 617-253-7038



The Koch Institute Proteomics Core Facility provides mass spectrometry-based analyses of protein samples for which one or more of the following is desired: Protein identification, protein characterization (including modifications), intact protein MW determination, protein quantitation. Samples which are not proteins or peptides, for which structural or MW information is needed, may also be submitted, as long as they are amenable to analysis with the instrumentation and methods currently available.

Protein Identification

  • Proteins separated by gel electrophoresis
    Protein identification from gel bands is generally routine and as long as there’s enough protein and the keratin contamination is kept under control it yields results. In general, Coomassie-stained bands contain enough protein to identify it, unless the keratin contamination is very high. A weakly stained Coomassie band may contain a 200-300 fmol protein (10s of ng) whereas a strongly-stained Coomassie band may contain pmol amounts of protein (100s of ng). Although the more protein there is the more confident the protein identifications will be, consider that by overloading a gel you may end up obscuring some of the weaker bands, especially if these are close to the bands of the major proteins.

    Silver-stained gel bands contain less protein, typically 100 fmol or less (low ng range) and the sensitivity of fluorescent stains is in-between that of Coomassie and silver. There are several staining kits available commercially from the major suppliers of biochemicals, which are compatible with mass spectrometric analysis (not all gel staining methods are). If you (or others in your lab) have submitted samples before and obtained useful results then obviously continue doing what you (or your colleagues) did before. Otherwise please talk to us before submitting samples so that we can help you increase the chances of success and avoid wasting time, effort and money.

    Issues to consider are keratin contamination and how to minimize it and the use of staining protocols that are compatible with mass spectrometric analysis. Keratin comes primarily from skin, wool clothing and dust. While handling samples and gels wear a lab coat and gloves (nitrile is said to be better than latex) and avoid touching samples, gels, etc. with bare hands. Rinse all containers, tubes, with HPLC-grade methanol and HPLC-grade water. If available, run the gel in a laminar flow hood, such as a tissue culture hood, if not available use a clean surface in an dust-free area (e.g. away from an air vent). After running and staining the gel place it in a rinsed Petri dish (avoid Ziplock bags) in 1-2% acetic acid, cover it and bring it over to our lab so we can cut the gel band(s) of interest in our laminar flow hood (if the timing doesn't work the gel can be stored overnight in the refrigerator in the covered Petri and this should not cause problems to its subsequent analysis). If you need to scan the gel ensure that the scanner surfaces have been rinsed with clean methanol and water and wiped clean. By the way, “old style” gel fixing (e.g. using glutaraldehyde or formaldehyde or similar protein cross-linking rteagents) must be avoided as it makes it impossible to digest proteins in gels and recover proteolytic peptides.

    For staining you can use any of the "mass spectrometry compatible" stains sold by several vendors. For Commassie, Gel Code Blue (Pierce/ThermoFisher) or Commassie Brilliant Blue R-250 and G-250 (Pierce or Bio-Rad) are all fine, if you follow the instructions that come with the staining kits. For silver, SilverQuest from Invitrogen or Silver Snap from Pierce are fine. Fluorescent gel stains (e.g. SYPRO Ruby, SYPRO Tangerine) are also mass spectrometry compatible. If you plan to use a stain and you cannot determine (e.g. from the product description) whether it is compatible with mass spectrometry please let us know and we can make inquiries.

    Although cutting gel bands is straight forward, it is important to do it in a clean area such as a laminar flow hood. If you have not done this before please talk to us before excising gel bands for analysis. We can arrange for you to bring your gels to our lab and we can cut gel bands you wish to identify.
    We prefer to carry out the in-gel digestions, under conditions that reduce sample contamination and optimize digestion and recovery of peptides, using protocols that have been applied successfully to thousands of samples. However we will accept already-digested samples, but if you have not submitted digested sample before please talk to us before doing this for the first time as we may be able to offer some helpful suggestions. We use trypsin for digestion; if you need your protein digested with another enzyme please discuss this with us.
  • Proteins in solution
    We routinely identify proteins in solutions, from a few to several hundreds. The same caveats apply as with in-gel digestion with regard to contamination (keratin is also a problem with solution samples, as proteolytic peptides from keratin often obscure or suppress signals from peptides from the less abundant proteins). Excessive amounts of salts can also be problematic, so using a method such as acetone precipitation to prepare protein samples may be necessary; please talk to us about this if you have not done this sort of thing before.
    In addition there are certain other materials and chemicals, such as detergents and polymers, which are much more problematic with solution samples compared to gel bands,. This is because excised gel bands are washed extensively prior to digestion so that many buffers and detergents can be removed without protein loss, but solution samples are not readily amenable to washing. Although methods such as MW filtration or dialysis can be used, they result in some protein loss and also some detergents and polymers stay with the protein anyway. Please talk to us regarding the use of detergents as we may be able to recommend alternative ways of preparing samples as well as some “mass spectrometry friendly” detergents.
    For on-gel protein sample it would be helpful to run a silver stained gel using ~10-15% of the sample; if you see bands, even weak ones, this means that there’s probably enough material in the sample remaining to identify some proteins.
  • Immunoprecipitation experiments
    Identifying proteins in immunoprecipitation experiments can be done in two ways: Either by running the immunoprecipitated proteins on a gel and submitting one or more gel bands or by submitting the mixture of immunoprecipitated proteins without separation. The same issues outlined above apply. Please note that if you run a silver stained gel using 10-15% of the immunoprecipitated sample and the only bands you see are related to the antibody used in your experiment it may not be possible to identify any of the other (and more relevant) proteins in your sample.

Protein Molecular Weight Measurement

We can measure protein molecular weights accurately, with errors in the range of 25-50 ppm (this is less than 3 Da for a 60 kDa protein) for single proteins. Protein mixtures also work but the data analysis (and the results generated) may get complicated because we do this by electrospray ionization, which generates many multiply charged ions for each protein and if there are several proteins in the sample the overlapping multiply charged ion envelopes may overwhelm the deconvoluation software. If you do not need the higher mass accuracy you may consider submitting your protein sample to the Biopolymers Core Facility for MALDI-TOF MS analysis, which would give you a MW with a mass error of 0.05-0.1% (30-60 Da for a 60 kDa protein), which is still much better than what you can estimate from SDS-PAGE.
Detergents should be avoided, as they suppress the ionization of protein samples and other contaminants such as polymers (PEG or PPG) have similar deleterious effects. However low-to-moderate concentration salt buffers (e.g. PBS, Tris) are fine, as protein samples are desalted on-line prior to MS analysis.
As with protein samples that are submitted for protein identification, please talk to us if what you need is something less straight-forward than simple MW determination, like the assessment of modifications such as phosphorylation or glyosylation or N- or C-terminus truncation. Depending on the heterogeneity of the sample and the MW the aforementioned modifications may or may not be discernible from the MW measurement of the intact protein and may require additional analysis of the proteolytic peptides obtained from digestion of the protein.

Protein Characterization

We can obtain detailed protein sequence information of relatively pure protein samples by digesting the protein and analyzing the resulting proteolytic peptides by LC-MS. The difference between protein characterization and protein identification is that in the former the identity of the protein is known and so is the amino acid sequence and the purpose of this analysis is to confirm the correctness of the sequence (e.g. of a point mutation, a splice variant or the presence of a tag) or identify specific known or suspected modifications to the amino acid sequence. For this it is important that as much of the protein sequence (e.g. 80% or more) is covered by identified proteolytic peptides, whereas a protein can be unambiguously identified from one or a few peptides that cover a mere 10-20% of the amino acid sequence. In general, for this type of work it is preferable that the protein sample be submitted in solution because in-gel digestion of proteins usually results in less than 50% sequence coverage (some parts of the protein embedded in the gel are not accessible to the enzyme and some of the peptides thus generated cannot be extracted from the gel – these problems generally do not arise when the protein is digested in solution). Typically larger amounts of protein (several pmol or 100s of ng) are needed for characterization (vs.10-50 times less for identification).
The aforementioned constraints notwithstanding, we will do our best to identify modifications in protein samples submitted for identification, whether in solution or as gel bands. We ask you to indicate on the sample submission form what type of modifications you are interested in and we will set up the database search software to look for the modifications indicated.

Protein Quantitation

We can provide estimates of protein amounts using label-free approaches as well as labeling methods such as SILAC and iTRAQ. For label-free quantitation the appropriate controls need to be run, for SILAC quantitation only certain isotopically labeled amino acid combinations are supported by the software we have available (you do the cell culture) and the iTRAQ chemistry, although relatively straight forward, requires following a specific protocol in detail. If you are interested in any of these methods please talk to us before generating samples for analysis in order to ensure that we are equipped to handle your request.

Analysis of Molecules other than Peptides and Proteins

We will analyze non-peptide samples as long as they behave like peptides. That is, they ionize in positive ion mass spectrometry, are water soluble and amenable to reversed phase HPLC analysis. Please note that the mass accuracy we can obtain for small molecule MW measurements (10-25 ppm) and the mass resolution (10,000-12,000) of our instrument may fall short of journal requirements for confirming the structure of a newly synthesized molecule. Also, since such samples are not regularly submitted for analysis, we may wait until enough small molecule samples have been submitted before setting up the instrument to analyze them. Therefore, wait times may be longer than usual for such analyses.

Instruments and Software

We have two mass spectrometers, an LTQ ion trap from Thermo and a QSTAR quadrupole-time-of-flight instrument from Applied Biosystems. Both are electrospray instruments and each is connected to a nanoflow HPLC. For the analysis of proteolytic peptides we use very small HPLC columns (50 or 75 mm internal diameter) at low flow rates (~300 nL/min), which increases sensitivity vis-à-vis analyses at higher flow rates. For identification of proteins we use either the SEQUEST database search software for the data generated with the LTQ instrument or the MASCOT database search software for the data generated with the QSTAR instrument (and sometimes with the LTQ data as well). We keep current with the various protein databases, with the help of the Bioinformatics Core Facility staff. For protein characterization and quantitation and MW determination we use software supplied with the instruments as well as third-party software. Please talk to us if you would like more information on our instrument and their capabilities of on the software we have available.

Contact Information

Please feel free to contact us if you have questions or to discuss specific items related to your projects.