Nanoelectrospray peptide mapping revisited: Composite survey spectra allow high dynamic range protein characterization without LCMS
International Journal of Mass Spectrometry Article in Press, Corrected Proof
Lu A, Waanders LF, Almeida R, Li G, Allen M, Cox J, Olsen JV, Bonaldi T, Mann M.
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.
http://dx.doi.org/10.1016/j.ijms.2007.05.006
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