Protein identification from database

D. Schleuder, F. Hillenkamp, and K. Strupat. IR-MALDI-Mass Analysis of Electroblotted Proteins Directly from the Membrane Comparison of Different Membranes, Application to On-membrane Digestion, and Protein Identification by Database Searching. Anal. Chem., 71(1999) 3238-3247. 

Manual protein identification from 2-DE gels is a time consuming technique that starts with spot excision and ends with the update of 2-DE databases. Nevertheless, this repetitive and exhaustive procedure could be mechanized using robots, thus increasing protein identification throughput. 

PMF, currently the most common method used to identify proteins in a high throughpnt environment, is based on the comparison of a list of experimental peptide masses with theoretical peptide masses. The experimental masses are generated from the MS measurement of an enzymatically digested protein sample. The theoretical masses are obtained from an in silico digestion of all sequences in a database. The goal is to find the protein(s) whose peptide masses show the best match with the experimental fingerprint. The method can be divided into 3 steps. The first step is pe detection, i.e. the selection of the most relevant masses for protein identification from the mass spectra. Frequently, only few experimental peptide... 

Peptide-mass fingerprinting with 2-DE-MALDI-TOF-MS. This is the most notable platform for proteome analysis [44,56,57]. The complex mixture of proteins is separated by 2-DE, the protein spots are cut from the gel, and proteins are subjected to in-gel digestion with trypsin. Salts are removed from the tryptic digest with a C-18 ZipTip. The peptides are eluted with the matrix solution and are deposited directly onto a 96-well MALDI plate. Mass measurement by TOF-MS provides peptide-mass fingerprints for subsequent protein identification with database search. 

The PepSeq program of Micromass s ProteinLynx software package was used for de novo analysis of the sequence (MS/MS) data. MS-Pattern of ProteinProspectror55 used the sequence tag determined from PepSeq to search the nonredundant database of the National Center for Biotechnology Information (NCBI) for protein identification. 

Fig. 6. Protein identification using a peptide map measured with a matrix-assisted laser desorption time-of-flight mass spectrometer. All the peptide extracted from the gel is measured and the set of masses is used in the database search. The mass resolution is in the order of 10,000. Individual isotopes of a 2.5 kDa peptide are clearly resolved.

Fig. 7. Protein identification with electrospray tandem mass spectrometry and a triple quadrupole mass spectrometer. Fragment spectra of several peptides are generated during one investigation. From the fragment spectra short sequence stretches can be read. Together with their mass location in the peptide of the measured mass, they can be used to specifically identify a protein in the database. Because the protein identification depends only on one peptide, several proteins can be identified from one sample.

Kinoshita K, Furui J, Nakamura H (2002) Identification of protein functions from a molecular surface database, eF-site. J Struct Funct Genomics 2 9-22... 

The basic identification process is analysis of the sequence or mass of six amino acids unique in the proteome of an organism, then to match it in a database. Put another way, protein identification is achieved converting proteins to peptides, determining the sequence of peptides, and then, matching with corresponding proteins from matching sequences in a database (Figure 5.3). 

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