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PMID: 16271296 Published · ppublish English Evaluation Study Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins.

Journal of the American Society for Mass Spectrometry ·Vol. 16 ·No. 12 ·2005-12-00 ·Pages 1985-99

Jebanathirajah JA, Pittman JL, Thomson BA, Budnik BA, Kaur P, Rape M, Kirschner M, Costello CE, O'Connor PB

Abstract

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.

MeSH Terms
Equipment Design Equipment Failure Analysis Peptide Mapping/methods Phosphopeptides/analysis,chemistry Phosphorylation Protein Processing, Post-Translational Proteins/analysis,chemistry Sequence Analysis, Protein/instrumentation,methods Spectrometry, Mass, Electrospray Ionization/instrumentation,methods Spectroscopy, Fourier Transform Infrared/instrumentation,methods
Chemicals
Phosphopeptides Proteins
Authors & Affiliations
9 authors, click to expand affiliations / ORCID
Jebanathirajah Judith A
Mass Spectrometry Resource, Department of Biochemistry, Boston University School of Medicine, MA 02115, USA.
Pittman Jason L
Thomson Bruce A
Budnik Bogdan A
Kaur Parminder
Rape Michael
Kirschner Marc
Costello Catherine E
O'Connor Peter B
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Article Info
Journal
Journal of the American Society for Mass Spectrometry
Abbr.
J Am Soc Mass Spectrom
ISSN
1044-0305
DOI
10.1016/j.jasms.2005.08.008
Published
2005-12-00
Epub
2005-00-02
Pages
1985-99
Language
English
Region
United States
NLM ID
9010412
Subset
IM
Grants
NHLBI NIH HHS · N01 HV28178 · United States
NCRR NIH HHS · P41 RR10888 · United States
External Links
PubMed source DOI: 10.1016/j.jasms.2005.08.008 View journal details
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