The Rockefeller University Proteomics Resource Center

Proteomics Information

Protein identification can be accomplished using three of the methods that we employ:

LC-MS/MS of proteolytic fragments of a protein coupled with database search (method of choice)
MALDI-TOF based peptide mass mapping of proteolytic fragments of a protein coupled with database search
Edman sequence analysis of intact protein or proteolytic fragments coupled with database search

Each of these methods has its advantages and disadvantages. You should carefully consider which of these methods is appropriate for your particular sample and situation before submitting your sample for analysis. Currently, the major method used at the Proteomics Resource Center is LC-MS/MS of proteolytic fragments of proteins from in gel or in solution digestion.

Sample preparation and sensitivity

All of these methods are suitable for analysis of Coomassie stained protein gel bands or spots. We have obtained nanogram-level detection of proteins with the Colloidal Blue Staining Kit (INVITROGEN). It is important to run and process the gel in as clean an environment as possible. Use thoroughly washed or disposable labware, wear gloves, and make sure reagents are fresh and uncontaminated.

You should clean a glass or plastic tray with detergent and rinse it thoroughly. You should wear clean, disposable gloves and never touch the gel bands directly. Never reuse stains and thoroughly acid wash staining trays if you reuse them.

The recommended Silver Staining Protocol.

The recommended in-gel digestion protocol.

For Edman sequencing analysis of intact protein, the protein must first be blotted to a PVDF membrane from the gel. This is not necessary, nor desirable, for any of these analyses following "in gel" proteolytic digestion of the protein band. In general, Edman sequencing is the least sensitive of the methods and will require a minimum of 1 picomol (preferably more) of protein to have a reasonable chance of success. The mass spectrometry based methods will routinely work with lower amounts of protein. Silver stained proteins are relatively difficult to analyze, even for moderately stained bands, and are not recommended.

Advantages and disadvantages of each method of analysis

LC-MS/MS: Nano HPLC coupled to an ion-trap or a QqTOF mass spectrometer. Analysis of proteolytic fragments.

High sensitivity (10 fmols)
Tandem mass spectrometry yields sequence dependent data
Several proteins can often be identified from a single sample
Possible to get very high confidence identifications due to sequence dependence of data
Exact, or high homology protein sequence must be present in database

MALDI-TOF based peptide mass mapping: Mass determination of proteolytic fragments.

High sensitivity
Relatively quick analysis
Relatively inexpensive
Requires relatively pure samples
More ambiguity in identifications due to lack of true sequence dependence of data
Exact, or high homology protein sequence must be present in database

Edman sequencing: Chemical sequence determination of intact, unblocked proteins or proteolytic fragments

Moderate sensitivity
Gives true de novo sequence data
Generally not affected by PTMs
Works with intact proteins, provided N-terminus is not blocked
Slowest of the three methods
Relatively expensive
Requires relatively pure samples (no more than one or two minor contaminants)
Analysis of proteolytic fragments requires chromatographic prefractionation

Click on the headings above for more detailed information.

In general, the LC-MS/MS method will yield the highest confidence identifications and is the method of choice provided the expected protein sequence or a highly homologous sequence is in the searched database. If you are dealing with an organism with a relatively small genome, MALDI-TOF peptide mass mapping analysis may be sufficient for your needs. If you are dealing with an organism whose genome is not known or you wish to obtain N-terminal sequence information on an intact protein, Edman sequencing analysis is probably your best choice since it yields sequence information.

Click here to see some examples of protein identification data obtained by these various methods.

Both Edman sequencing and LC-MS/MS are useful for looking at protein modifications such as phosphorylations, acetylations, methylations, etc. However, due to the relatively low resolution characteristics of our ion-trap instrument, unambiguous assignments of modification sites can be difficult with this instrument. Edman sequencing can offer more unambiguous identification of modification sites, provided that suitable standards for the modified amino acids are available. Edman sequencing also suffers from the limitations listed above. A combination of these two techniques can often yield unambiguous identification and location of some modifications (click to see an example. You will need Adobe Acrobat Reader).

This page was last updated on 4/27/07

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