Peptide mass profile

Computer algorithms facilitate identification of the open reading frames that encode a given protein by using partial sequences and peptide mass profiling to search sequence databases. 

Gharahdaghi F, Kirchner M, Fernandez J, Mische SM. Peptide-mass profiles of polyvinylidene difluoride-bound proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence of nonionic detergents. Anal Biochem 1996 233 94-9. 

Figure 6. Peptide mass profiling of a silver stained protein spot from a narrow range, pH 4-7, 2DE separation of human heart (ventricle) proteins. A MALDI-TOF mass spectrum of tryptic peptides is shown, analyzed using a Micromass Tofspec 2E spectrometer (Manchester, UK) operated in the positive ion reflectron mode at 20 kV accelerating voltage with time-lag focusing enabled. The protein spot of interest was identified as human vimentin, (courtesy of J.A.Westbrook and R. Wait) (unpublished data). 2DE gels were silver stained using a modified Amersham Biosciences kit.

A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. 

The MALDI-TOF-MS platform is able to perform high-throughput quantitative peptide profiling using biofluids, in parhcular serum, which is highly relevant for chnical use. It has been shown that semm and CSF peptide mass profiles can be correlated to clinical data for disease signature discovery and validation. 

The most widely used mass spectrometric identification procedure is MALDI-Tof analysis of the entire peptide mixture. Gas-phase matrix interaction with peptide ions in MALDI-Tof results in singly charged ions, giving a mass profile that is highly characteristic of the protein from which the peptides are derived. These peptide masses (actually protonated peptide molecular ions, MH+) can be used to search databases (either protein or nucleic acid databases) to identify the proteins. The two most important factors in successfully identifying proteins by this approach are the number of matching peptide masses and the accuracy of the peptide mass determination. 

The profile of the masses of the peptides obtained by one of these methods is compared by means of a computer with all predicted peptide digests from a database of proteins to identify the best possible matches. This is termed peptide mass fingerprinting (PMF). A protein generally can be identified using the mass of four to six of its cleavage peptides having masses in the range 700-3000 Da and determined with an accuracy of 0.05 to 0.0005 %. This is improved further if the molecular mass of the protein also is provided [83-86],... 

The mass profile is then compared with peptide masses predicted from theoretical digestion of known protein sequences contained within current protein databases or predicted from nucleotide sequence databases. This approach proves very effective when trying to identify proteins from species... 

Another example of the use of mass spectrometry to delect toxins is to identify ricin, a highly toxic protein that inhibits cell protein synthesis. Ricin is produced from the seeds of Ricinus communis plants (known conunonly as castor beans) [73]. Structurally, ricin is made of A- and B-chains linked by a disulfide bridge. The toxicity of ricin is due primarily to the A-chain, which acts as an RNA A-glycosidase, which leads to ribosome incapacitation and ultimately to cell death. Ricin was identified from crude castor bean extracts using LC-MS/MS. The extract was denatured, reduced, and alkylated prior to trypsin digestion. Ricin identification was based on the detection of marker peptides in the digest. These markers include T5, T7, Til, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. MS/MS can provide the amount and sequence of each marker for irrefutable evidence. For quick screening of ricin in crude extracts, MALDI-MS can be used to provide the molecular mass profile of the marker peptides. 

---