How many proteins can be identified from a single gel band?

This depends on the complexity of the sample that was loaded on the gel. From our experience we have identified as many as 70 proteins from a single gel band when complex cell lysate was ran on 12%T minigel. We strongly recommend not to overload the gel as it will affect the resolution and separation of proteins. Moreover, it can result in “carry-over” of abundant peptides in LC resulting in identification of same protein repeatedly in different samples. 

Can I submit a silver-stained gel?

Yes, if it is stained by mass spectrometry-compatible silver stain protocol. Many commercial vendors sell such kits.

>Publications

Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM “Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity.”   Electrophoresis, 1999, 20(3)   p. 601-5.   Summary |    Full Text (DOI)

Aurandt J, Vikis HG, Gutkind JS, Ahn N, Guan KL “The semaphorin receptor plexin-B1 signals through a direct interaction with the Rho-specific nucleotide exchange factor, LARG.”   Proc Natl Acad Sci U S A, 2002, 99(19)   p. 12085-90.   Summary |    Full Text (PMC) |    Full Text (DOI)

>Web Links

Life Technologies SilverQuest  Pierce Silver Stain Kit  Sigma ProteoSilver

Which stains (other than Coomassie blue) are compatible with mass spectrometry?

Negative staining techniques such as zinc stain are compatible with mass spectrometry. Many of the fluorescent stains are also compatible with mass spectrometry.

>Related Questions

Can I submit a silver-stained gel?

>Web Links

Sypro Ruby  Colloidal Coomassie Blue  Flamingo

Do you use only trypsin to digest proteins or can other proteolytic enzymes be used?

If sample is to be “in-solution digested,” we can use any one or combination of commercially available sequencing-grade proteolytic enzymes. For “in-gel digestion”, the repertoire of the proteolytic enzymes becomes slightly more limited. Note that many high molecular weight enzymes are not as efficient for in-gel digestion as trypsin and chymotrypsin.

>Related Questions

How many proteins can be identified from a single gel band?

>Web Links

Promega Trypsin  Roche Lys-C  Promega Chymotrypsin  Roche Asp-N

I have detergent in my sample. Are there ways to remove it?

Yes. The simplest and cheapest way is to use precipitation technique like TCA and acetone precipitation. The downside of these methods is that once precipitated, some proteins are hard to resuspend in the buffer of choice. At   Northwestern Proteomics, we offer a service to remove the detergent using Pierce detergent removal kit. We have successfully used this kit for removal of octyl maltosides and SDS from the sample. It should be noted that success of all any technique will depend on concentration and type of the detergent.

>Web Links

HiPPR Detergent Removal Resin

>Protocols

DOWNLOAD

I have my protein complex in a buffer with very high salt concentration plus 5% glycerol. Is this buffer compatible with mass spectrometry?

No. Salts and glycerol are not compatible with mass spectrometry. If the samples contain salts or glycerol, we will desalt the sample either by using a zip tip or C18 spin columns.

Can I use in-house Coomassie blue stain? Is there a preferred protocol ?

You can use in house made stain. But note that presence of acetic acid and methanol can modify proteins. We recommend to use commercially available Coomassie blue stains as they are solvent free and are more sensitive than conventional staining. We do not have any preferred commercial vendors.

>Publications

Haebel S, Albrecht T, Sparbier K, Walden P, Körner R, Steup M “Electrophoresis-related protein modification: alkylation of carboxy residues revealed by mass spectrometry.”   Electrophoresis, 1998, 19(5)   p. 679-86.   Summary |    Full Text (DOI)

Are there any good papers describing phosphoproteomics?

Yes! See the publication linked here.

>Publications

Dephoure N, Gould KL, Gygi SP, Kellogg DR “Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.” Mol Biol Cell  , 2013, 24(5) p. 535-42.   Summary |    Full Text (PMC) |    Full Text (DOI)

How do I submit a sample to be run?

Information on sample submission can be found   here.

What search engine do you use?

We use Mascot (Matrix Science) for protein identification. Protein inference, grouping and FDR (false discovery rate) estimation is performed in Proteome Discoverer and Scaffold.

>Publications

Perkins DN, Pappin DJ, Creasy DM, Cottrell JS “Probability-based protein identification by searching sequence databases using mass spectrometry data.” Electrophoresis , 1999, 20(18) p. 3551-67.  Summary |   Full Text (DOI)

>Web Links

Mascot Scaffold

What database is used for the searches? Are databases updated regularly?

Our default database is UniProt. As routine policy, for poorly annotated proteomes we search both the sections of Uniprot i.e. SwissProt (curated) and Trembl (non-curated). After both the searches, non redundant protein list with each IDs supported by at least one unique peptide is submitted to the user. Both databases are updated every month.

What is a false discovery rate (FDR) ?

The reporting of a FDR has become mandatory. The FDR is essentially a statistical and computational measure to establish the certainty of the protein identification in a given dataset. There are several methods, we typically use a decoy database to measure the false discovery rate.

>Publications

Elias JE, Gygi SP “Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.” Nat Methods , 2007, 4(3) p. 207-14.  Summary |   Full Text (DOI)

Keller A, Nesvizhskii AI, Kolker E, Aebersold R “Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.”  Anal Chem, 2002, 74(20)  p. 5383-92.  Summary |   Full Text (DOI)

Are there any good papers describing phosphoproteomics?

Yes! See the publication linked here.

>Publications

Dephoure N, Gould KL, Gygi SP, Kellogg DR “Mapping and analysis of phosphorylation sites: a quick guide for cell biologists.” Mol Biol Cell , 2013, 24(5) p. 535-42.  Summary |   Full Text (PMC) |   Full Text (DOI)