TY - JOUR
T1 - Nanoelectrospray peptide mapping revisited
T2 - Composite survey spectra allow high dynamic range protein characterization without LCMS on an orbitrap mass spectrometer
AU - Lu, Aiping
AU - Waanders, Leonie F.
AU - Almeida, Reinaldo
AU - Li, Guoqing
AU - Allen, Mark
AU - Cox, Jürgen
AU - Olsen, Jesper V.
AU - Bonaldi, Tiziana
AU - Mann, Matthias
PY - 2007/12/1
Y1 - 2007/12/1
N2 - 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.
AB - 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.
KW - Dynamic range
KW - LTQ-Orbitrap
KW - Peptide mass mapping
KW - Peptide sequencing
KW - Protein modification
UR - http://www.scopus.com/inward/record.url?scp=35748939071&partnerID=8YFLogxK
U2 - 10.1016/j.ijms.2007.05.006
DO - 10.1016/j.ijms.2007.05.006
M3 - Journal article
AN - SCOPUS:35748939071
SN - 1387-3806
VL - 268
SP - 158
EP - 167
JO - International Journal of Mass Spectrometry
JF - International Journal of Mass Spectrometry
IS - 2-3
ER -