Complete characterization of posttranslational modification sites in the bovine milk protein PP3 by tandem mass spectrometry with electron capture dissociation as the last stage.
A comprehensive approach to protein identification and determination of sites of posttranslational modifications (PTMs) in heavily modified proteins was tested. In this approach, termed "reconstructed molecular mass analysis" (REMMA), the molecular mass distribution of the intact protein is measured first, which reveals the extent and heterogeneity of modifications. Then the protein is digested with one or several enzymes, with peptides separated by reversed-phase HPLC, and analyzed by Fourier transform mass spectrometry (FTMS). Vibrational excitation (collisional or infrared) or electron capture dissociation (ECD) of peptide ions provides protein identification. When a measured peptide molecular mass indicates the possibility of a PTM, vibrational excitation is applied to determine via characteristic losses the type and eventually the structure of the modification, while ECD determines the PTM site. Chromatographic peak analysis continues until full sequence coverage is obtained, after which the molecular mass is reconstructed and compared with the measured value. An agreement indicates that the PTM characterization was complete. This procedure applied to the bovine milk PP3 protein containing 25% modifications by weight yielded all known modifications (five phosphorylations, two O- and one N-glycosylation) as well as the previously unreported NeuNAc-Hex-[NeuNAc]HexNAc group O-linked to Ser60. With the FTMS performance improved, REMMA can serve as the basis for high-throughput, high-sensitivity PTM characterization of biological important proteins, which should speed up the proteomics research.
Kjeldsen
Frank
F
University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Haselmann
Kim F
KF
Budnik
Bogdan A
BA
Sørensen
Esben S
ES
Zubarev
Roman A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005583:Fourier Analysis; D006031:Glycosylation; D008894:Milk Proteins / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D008894:Milk Proteins; D010446:Peptide Fragments