Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.
The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.
Molloy
M P
MP
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Andrews
P C
PC
D000818:Animals; D006801:Humans; D010748:Phosphopeptides / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis*; D010769:Phosphothreonine / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling; D013438:Sulfhydryl Compounds / Q000737:chemistry
D010748:Phosphopeptides; D010750:Phosphoproteins; D013438:Sulfhydryl Compounds; D010769:Phosphothreonine; D010768:Phosphoserine