Ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry and tandem mass spectrometry for peptide de novo amino acid sequencing for a seven-protein mixture by paired single-residue transposed Lys-N and Lys-C digestion.
BACKGROUND
Bottom-up tandem mass spectrometry (MS/MS) is regularly used in proteomics to identify proteins from a sequence database. De novo sequencing is also available for sequencing peptides with relatively short sequence lengths. We recently showed that paired Lys-C and Lys-N proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Such parallel experiments provide complementary information, and allow for up to 100% MS/MS sequence coverage.


METHODS
Here, we report digestion by paired Lys-C and Lys-N proteases of a seven-protein mixture: human hemoglobin alpha, bovine carbonic anhydrase 2, horse skeletal muscle myoglobin, hen egg white lysozyme, bovine pancreatic ribonuclease, bovine rhodanese, and bovine serum albumin, followed by reversed-phase nanoflow liquid chromatography, collision-induced dissociation, and 14.5 T Fourier transform ion cyclotron resonance mass spectrometry.


RESULTS
Matched pairs of product peptide ions of equal precursor mass and similar retention times from each digestion are compared, leveraging single-residue transposed information with independent interferences to confidently identify fragment ion types, residues, and peptides. Selected pairs of product ion mass spectra for de novo sequenced protein segments from each member of the mixture are presented.


CONCLUSIONS
Pairs of the transposed product ions as well as complementary information from the parallel experiments allow for both high MS/MS coverage for long peptide sequences and high confidence in the amino acid identification. Moreover, the parallel experiments in the de novo sequencing reduce false-positive matches of product ions from the single-residue transposed peptides from the same segment, and thereby further improve the confidence in protein identification. Copyright © 2016 John Wiley & Sons, Ltd.
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL, 32310, USA.
Xiaoyan
X
Guan
Department of Behavioral Sciences and Social Medicine, College of Medicine, Florida State University, 1115 W. Call St., Tallahassee, FL, 32306, USA.|Department of Statistics, Florida State University, 117 N. Woodward Ave., Tallahassee, FL, 32306, USA.
Naomi C
http://orcid.org/0000-0002-9991-427X
NC
Brownstein
Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine, One Baylor Plaza, MS-125, Houston, TX, 77030-3411, USA.
Nicolas L
NL
Young
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, FL, 32310, USA.|Department of Chemistry and Biochemistry, Florida State University, 95 Chieftain Way, Tallahassee, FL, 32303, USA.
Alan G
AG
Marshall
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D010446:Peptide Fragments; D011506:Proteins; D010447:Peptide Hydrolases