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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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