LC-MS/MS analysis of peptides with methanol as organic modifier

Improved limits of detection

Francesco Giorgianni, Achille Cappiello, Sarka Beranova, Pierangela Palma, Helga Trufelli, Dominic M. Desiderio

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

With the advent of soft ionization methods such as MALDI and ESI, mass spectrometry has become the most important technique for the analysis of proteins and peptides. ESI-MS is often preceded by separation of the peptide sample by reversed-phase liquid chromatography (LC). Acetonitrile (ACN) is the most commonly employed organic solvent in LC-ESI-MS analysis of peptides. In this report, we demonstrate that the use of methanol (MeOH) as the organic modifier improves the detection limits for analysis of peptide mixtures such as those found in tryptic digests of proteins. A nanoLC-ESI-quadrapole ion trap instrument (LCQ Deca, ThermoFinnigan) was used to analyze peptide standards, protein digests of known concentrations, and tryptic digests of 2-DGE-separated proteins. MeOH displayed excellent chromatographic performance (separation and sensitivity), and shorter gradient times were possible for chromatographic separation with MeOH versus ACN. Sensitivity levels of a few hundred attomoles were achieved with MeOH; those levels could not be achieved with ACN. In addition, MeOH-based nanoLC-MS/MS yielded superior results for the analysis of digests of 2-DGE-separated proteins. For the 14 protein spots analyzed, the success rate of protein identification with MeOH-based nanoLC-ESI-MS/MS was 100%, with multiple proteins identified in several of the spots. In contrast, ACN-based procedure failed to identify any proteins in 21% of the spots and overall identified 33% fewer proteins than the MeOH-based procedure. In summary, higher sensitivity and shorter gradient times make MeOH an excellent organic modifier for the use in nanoLC-ESI-MS/MS analysis of peptides.

Original languageEnglish (US)
Pages (from-to)7028-7038
Number of pages11
JournalAnalytical Chemistry
Volume76
Issue number23
DOIs
StatePublished - Jan 3 2005

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Liquid chromatography
Methanol
Peptides
Proteins
Organic solvents
Ionization
Mass spectrometry
Ions
acetonitrile

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

LC-MS/MS analysis of peptides with methanol as organic modifier : Improved limits of detection. / Giorgianni, Francesco; Cappiello, Achille; Beranova, Sarka; Palma, Pierangela; Trufelli, Helga; Desiderio, Dominic M.

In: Analytical Chemistry, Vol. 76, No. 23, 03.01.2005, p. 7028-7038.

Research output: Contribution to journalArticle

Giorgianni, Francesco ; Cappiello, Achille ; Beranova, Sarka ; Palma, Pierangela ; Trufelli, Helga ; Desiderio, Dominic M. / LC-MS/MS analysis of peptides with methanol as organic modifier : Improved limits of detection. In: Analytical Chemistry. 2005 ; Vol. 76, No. 23. pp. 7028-7038.
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