Tandem mass spectrometry for structural characterization of proline-rich proteins: application to salivary PRP-3.
Proline-rich proteins (PRPs), including collagens, complement 1q, and salivary PRPs, are unusually difficult to sequence by mass spectrometry, due to the high efficiency of cleavage at the amide bond on the N-terminal of proline residues and the consequently low relative abundance of fragment arising from cleavages at other amide bonds. To fully characterize these proteins by mass spectrometry, specialized approaches to fragmentation are needed for the peptides with high proline content. Our work reported herein focused on the analysis of the set of peptides derived by tryptic cleavage of the salivary protein PRP-3. Two methods of fragmentation were compared: Collision-induced dissociation (CID) and electron capture dissociation (ECD). The data obtained demonstrated that ECD spectra of peptides containing more than 30% proline residues are simpler and easier to interpret than are CID spectra of those peptides. Factors that limit the two methods of fragmentation include the complexity of information contained in the CID spectra and the low efficiency of ECD processes. A complementary approach using both decomposition methods provides more complete and interpretable sequence information and yielded >93% sequence coverage for the 11-kDa PRP-3 isolated from human saliva.
Leymarie
Nancy
N
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Berg
Eric A
EA
McComb
Mark E
ME
O'Connor
Peter B
PB
Grogan
James
J
Oppenheim
Frank G
FG
Costello
Catherine E
CE
D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011392:Proline; D055232:Proline-Rich Protein Domains; D012463:Saliva / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010455:Peptides; D012471:Salivary Proteins and Peptides; D011392:Proline; D014357:Trypsin