A simple and efficient approach to improve protein identification by the peptide mass fingerprinting method: Concomitant use of negative ionization

Takao Sanaki, Mao Suzuki, Seon Hwa Lee, Takaaki Goto, Tomoyuki Oe

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

Peptide mass fingerprinting (PMF) has been widely used as an efficient analytical strategy for protein identification. This is most commonly used with a combination of protein digestion using sequence-specific proteases and MALDI-TOFMS. Then database searches are performed comparing the pattern of the experimentally obtained masses with the pattern of the theoretical peptide masses of proteins stored in the database. The positive ionization mode has been mainly used for MALDI analyses with a few exceptions for phosphopeptides, oligonucleotides, etc. Therefore, nonphosphorylated peptides that have low pI values could be missed from PMF using the positive ionization mode. Here, we introduce optimal conditions for negative ionization of peptides and the practical advantages of negative ionizations in PMF. Angiotensin I (pI 6.9) and bovine serum albumin (BSA) tryptic digests were used as model peptides. Eight matrix candidates and seven additives were examined in terms of sensitivity, robustness and reproducibility. The combination of DHB and phosphoric acid was the best condition for negative ionization of peptides and was found to be compatible with the positive ionization mode. Using 150 mM DHB/1% phosphoric acid, the coverage (% by amino acid count) of BSA tryptic digest (0.6 pmol per spot) totaled 67.2% (negative + positive). The 24.1% of peptides (pI range 4.1-6.2) were detected only by negative ionization, which indicated that acidic peptides were efficiently recovered by the negative ion mode. This methodology has been successfully employed to analyze other proteins without false positive identifications.

Original languageEnglish
Pages (from-to)1144-1151
Number of pages8
JournalAnalytical Methods
Volume2
Issue number8
DOIs
Publication statusPublished - 2010 Aug

ASJC Scopus subject areas

  • Analytical Chemistry
  • Chemical Engineering(all)
  • Engineering(all)

Fingerprint Dive into the research topics of 'A simple and efficient approach to improve protein identification by the peptide mass fingerprinting method: Concomitant use of negative ionization'. Together they form a unique fingerprint.

Cite this