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Defining Intact Protein Primary Structures from Saliva: A Step toward the Human Proteome Project

Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was...

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Published in:Analytical chemistry (Washington) 2012-05, Vol.84 (10), p.4383-4395
Main Authors: Halgand, F, Zabrouskov, V, Bassilian, S, Souda, P, Loo, J. A, Faull, K. F, Wong, D. T, Whitelegge, J. P
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description Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved, and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time-of-flight instrument to quickly identify and characterize proteins, and data acquisition was switched to Fourier-transform ion cyclotron resonance (FTICR) for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-mass spectrometry (MS) instrument combined with electron-capture dissociation (ECD) provided the most informative data sets, with the more frequent presence of “unique” ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS data sets that allow researchers to completely assign the identity and the structure of a protein.
doi_str_mv 10.1021/ac203337s
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subjects Amino Acid Sequence
Analytical chemistry
Body fluids
Chemistry
Chromatography, High Pressure Liquid
Exact sciences and technology
General, instrumentation
Histatins - chemistry
Humans
Mass spectrometry
Molecular Sequence Data
Molecular structure
Peptides
Proteins
Proteins - chemistry
Proteome
Proteomics
Saliva - metabolism
Spectrometric and optical methods
Spectrometry, Mass, Electrospray Ionization
title Defining Intact Protein Primary Structures from Saliva: A Step toward the Human Proteome Project
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