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Reputable Mentor II
Reputable Mentor II
Lu A, Waanders LF, Almeida R, Li G, Allen M, Cox J, V. Olsen JV, Bonaldi T, Mann M.
Int. J Mass Spectrom. 2007 Dec;268:158-167.
Mass spectrometric (MS) determination of the primary structure of proteins, including post-translational modifications, remains a challenging task. Proteins are usually digested to tryptic peptides that are measured either by MALDI peptide mapping or by liquid chromatography online coupled to tandem MS (LC–MS/MS). Here we instead analyze peptides by a chip implementation of nanoelectrospray (TriVersa Nanomate, Advion Biosciences), coupled to a linear ion-trap–orbitrap hybrid instrument (LTQ-Orbitrap, Thermo Fisher). The C-trap connecting the linear ion-trap and orbitrap is filled repeatedly in different m/z ranges with up to a million charges. Each range is analyzed in the orbitrap repeatedly and separately, creating a survey spectrum composed of hundreds of single spectra. The composite spectrum is inherently normalized for different m/z ranges due to their different fill times and retains information on the variability of mass measurement and intensity. Nanoelectrospray offers analysis times of more than 30 min/μl of peptide mixture, sufficient for in-depth peptide characterization by high resolution C-trap fragmentation in addition to high sensitivity ion-trap fragment analysis. We obtain over 6000-fold dynamic range and subfemtomole sensitivity. Automated analysis of digested BSA resulted in sequence coverage above 80% in low femtomole amounts. We also demonstrate identification of seven modified peptides for a purified histone H3 sample. Static spray allows relative quantitation of the same peptide with different modifications. Chip-based nanoelectrospray on an orbitrap instrument thus allows very high confidence protein identification and modification mapping and is an alternative to MALDI peptide mapping and LC–MS/MS.
Department for Proteomics and Signal Transduction, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18, D-82131 Martinsried, Germany
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