Charting the Proteomes of Organisms with Unsequenced Genomes by MALDI-Quadrupole Time-of-Flight Mass Spectrometry and BLAST Homology Searching

Abstract

MALDI-quadrupole time-of-flight mass spectrometry was applied to identify proteins from organisms whose genomes are still unknown. The identification was carried out by successively searching a sequence database-first with a peptide mass fingerprint, then with a packet of noninterpreted MS/MS spectra, and finally with peptide sequences obtained by automated interpretation of the MS/MS spectra. A "MS BLAST" homology searching protocol was developed to overcome specific limitations imposed by mass spectrometric data, such as the limited accuracy of de novo sequence predictions. This approach was tested in a small-scale proteomic project involving the identification of 15 bands of gel-separated proteins from the methylotrophic yeast Pichia pastoris, whose genome has not yet been sequenced and which is only distantly related to other fungi.

Expanding the organismal scope of proteomics: Cross‐species protein identification by mass spectrometry and its implications

“…However, the absence of a rigorous scoring system may lead to erroneous identifications as the correct sequence may be present in the list, but may not be ranked among the top hits. Even though it is difficult to use these sequences for cloning (where the requirement is that the sequences should be long, 100% accurate, and encode for low degeneracy primers), they can be successfully used for identifying proteins in a sequence database using various sequence similarity search algorithms [23][24][25].…”

“…Bovine DNA polymerase was the top hit. Multiple hits from different organisms were retrieved from the MS BLAST search and many were able to make high confidence matches (see Table 1, a)) available as stand-alone applications [23,28,29], whereas MS BLAST (Mass Spectrometry driven BLAST) [25] is accessible over the web [30]. The limitations of FASTA-based algorithms is that they are slow search engines and the final score of hits depends not only on the number of matched peptides, but decreases with the number of peptide sequences submitted in a query.…”

“…The MS BLAST software has a particular advantage of being very fast in searching and not penalizing the score of hits for submissions of numerous redundant putative sequence candidates (Fig. 3) [25]. This allows direct submission of the entire output of the sequence prediction software for all fragmented peptides, without intermediate inspection of data and arbitrary selection of the most reliable hits.…”

“…However, using MS and advanced methods of database interrogation, it is becoming increasingly possible to study the proteomes of organisms with unsequenced genomes. Cross-species identifications have been made in these organisms and others not cited: Zea mays [46,47], Pisum sativum [48,49], Papaver somniferum [50], Spinacia oleracea [44], Arabidopsis thaliana [44], Bos taurus [51], Xenopus laevis [30,52], Pichia pastoris [25], and Trypanosoma brucei [28,29]. Many earlier studies utilizing cross-species identification of unknown proteins relied on high mass accuracy MALDI peptide mapping, and therefore may have identified proteins or enzymes highly conserved across the biosphere.…”

Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-Trimethoxyphenyl) phosphonium: Implications for the aspartic acid cleavage mechanism

“…A partial ion series also can be used in conjunction with database searching. In this mass tag scheme, a partial sequence and the peptide molecular weight are matched to the correct sequence from a database [13][14][15]. Identified peptides are matched against theoretical protein digests to identify the protein.…”