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Proteomics: 2D PAGE & Mass Spectrometry

 Over the last 2 years an extremely powerful combination of 2D gel separation and mass analysis has emerged and is now complementing genome projects world-wide. The essence of this approach is to use 2D PAGE to separate proteins from a specific cell or tissue type, record the pattern, then produce a Western Blot. The blot can either be stained or visualised by a variety of methods to detect protein or simply divided up into regions. Proteins in the blot are digested with a proteolytic enzyme (such as trypsin) which has a well-defined cleavage specificity. Peptide fragments are eluted and analysed (frequently using Matrix-Assisted Laser Desorption Mass Spectroscopy - MALDI-MS, which has femtomole sensitivity).

The resulting peptide masses (which will be accurate to within 1 Dalton) are then compared with theoretical masses calculated from amino-acid sequence data bases. Generally a good mass match for around 5 peptides is quite sufficient to pinpoint the identity of an unknown protein. Even if the protein is not in the sequence databases, if an internal portion of it is homologous (has very similar sequence to) a protein that is known, then it may be picked up and an hypothetical function assigned on the basis of mass-mapping alone!

To find out more about this powerful adjunct to 2D Page, the following links will be helpful:

SWISS-PROT Peptide Mass Search SWISS PROT ExPaSY or EMBL Protein databases using peptide masses from specific enzyme digestion. Can unambiguously identify proteins from 5-7 peptide masses alone.
FindMod FindMod is a tool that can predict potential protein post-translational modifications and find potential single amino acid substitutions in peptides.
Prot-ID Protein identity from enzymatic fragments (Rockefeller)
Peptide-mass PEPTIDE MASS SEARCH searches the protein sequence database for multiple peptides of individual proteins that match the measured masses determined by mass spectrometry. The protein database in FASTA-style includes SwissProt, Protein Identification Resource (PIR) and SwNew. It does not contain fragments. The program FRAGFIT (1) was modified to compute the masses with some posttranslational modifications (2).
Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein databases, PNAS 90:5011-5015
Thiede B, Otto A, Zimny-Arndt U, Mueller EC, and Jungblut P (1996) Identification of human myocardial proteins separated by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry, Electrophoresis 1996, 17:588-599
Ms-fit UCSF peptide mass fingerprinting tool
Ms-Fragment ion tag Search software offered by UCSF
NYC-MASS Protein mass spectometry database (Rockefeller)
Murray Murray's Collection of Links to Mass Spectrometry Internet Sites
EMBL EMBL Bio Mass Spectrometry Software Archive

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Proteomics meetings

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