Iterative data analysis is the key for exhaustive analysis of peptide mass fingerprints from proteins separated by two-dimensional electrophoresis

Abstract

Peptide mass fingerprinting (PMF) is a powerful tool for identification of proteins separated by two-dimensional electrophoresis (2-DE). With the increase in sensitivity of peptide mass determination it becomes obvious that even spots looking well separated on a 2-DE gel may consist of several proteins. As a result the number of mass peaks in PMFs increased dramatically leaving many unassigned after a first database search. A number of these are caused by experiment-specific contaminants or by neighbor spots, as well as by additional proteins or post-translational modifications. To understand the complete protein composition of a spot we suggest an iterative procedure based on large numbers of PMFs, exemplified by PMFs of 480 Helicobacter pylori protein spots. Three key iterations were applied: (1) Elimination of contaminant mass peaks determined by MS-Screener (a software developed for this purpose) followed by reanalysis; (2) neighbor spot mass peak determination by cluster analysis, elimination from the peak list and repeated search; (3) re-evaluation of contaminant peaks. The quality of the identification was improved and spots previously unidentified were assigned to proteins. Eight additional spots were identified with this procedure, increasing the total number of identified spots to 455. [5][6][7], represent major milestones on the way to an understanding of the proteome. A further step was the application of mass spectrometry for peptide mass fingerprinting (PMF) instead of SDS gel electrophoresis [8], which resulted in a quick and reliable identification of 2-DE separated protein spots [9,10]. Instead of measuring the mass of the uncleaved proteins the high mass accuracy of MALDI-and ESI-MS in the Mr range between 500 and 3000 was utilized.PMF as a probability method is especially powerful if the data set of potential protein candidates is small, as in the case of microorganisms with completely sequenced genomes. For unequivocal identification of post-translational modifications, complementation by protein sequencing methods is necessary. PMF has been used successfully for the identification of several hundred proteins of Haemophilus influenzae [11], Mycobacterium tuberculosis [12], Helicobacter pylori [13], and Mycoplasma pneumoniae [14]. These investigations clearly demonstrated that proteins often occur as different protein species at different positions within the 2-DE gel. However, one spot may also represent several

Analysis of Automatically Generated Peptide Mass Fingerprints of Cellular Proteins and Antigens from Helicobacter pylori 26695 Separated by Two-dimensional Electrophoresis

“…Improvement of Identification, the Search for Spot Contaminants, and Additional Spot Components-To improve identification and detect minor spot components in large datasets we developed the iterative data analysis software MSScreener (16). The new version integrates tools for contaminant search and removal, calculation and plotting of the decimal places of masses, and calculation of interval matrices to perform clusterings in R (Fig.…”

“…2). Here a threshold of 5% (mass 16). Parameters for automatic and manual data were 30 and 100 ppm, respectively, and fixed and variable methionine oxidations, respectively.…”

“…Now the question is if the information content of the remaining peaks is sufficient to identify minor components of spots, which could contribute to a higher coverage of the proteome by the 2-DE/MALDI-MS approach. In a first report we demonstrated the application of a software program named MS-Screener together with cluster analysis starting with an H. pylori dataset of 480 PMFs obtained by manual spot picking, digestion, and peak detection (16).…”

Shifting unoccupied spectral space in mass spectrum of peptide fragment ions

“…The average fractional mass (⌬m) of intact peptides increases with the monoisotopic mass (m), according to the following equation [4]. Similar equations have also been presented previously [1,3,7]. ⌬m ϭ 4.7831 ϫ 10 Ϫ4 ϫ m ϩ 0.0279…”

“…Rescaling the residue mass to the averagine scale basically eliminates the systematic drifting in the mass defect of peptide ions, which increases with the residue mass on the IUPAC scale [1,3,4,7].…”