Distinguishing between phosphorylated and nonphosphorylated peptides with ion mobility-mass spectrometry.
Mass spectrometry has become an indispensable tool in identifying post-translationally modified proteins, but multiple peptide mass-mapping/peptide-sequencing experiments are required to answer questions involving the site and type of modification present. Here, we apply ion mobility-mass spectrometry (IM-MS), a high-throughput analysis method having high selectivity and sensitivity, to the challenge of identifying phosphorylated peptides. Ion mobility separation is based on the collision cross-section of the ion. Phosphorylation can result in a conformational change in gas-phase peptide ions, which can be detected by IM. To demonstrate this point, a peptide mixture containing a variety of peptide sequences is examined with IM-MS and molecular dynamics calculations. During the course of these studies, two classes of phosphopeptide were identified: (i) phosphorylated peptide ions that have conformers that differ from the nonphosphorylated ion and (ii) phosphorylated peptide ions that have conformations that are very similar to the nonphosphorylated peptide. The utility of IM-MS peptide mass mapping for identifying both types of phosphorylated peptides is discussed.
Ruotolo
Brandon T
BT
Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.
Verbeck
Guido F
GF
Thomson
Lisa M
LM
Woods
Amina S
AS
Gillig
Kent J
KJ
Russell
David H
DH
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002364:Caseins; D010455:Peptides; D010748:Phosphopeptides; D000804:Angiotensin II