Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis.
A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, United States.
Suzanne E
SE
Kulevich
Brian L
BL
Frey
Gloria
G
Kreitinger
Lloyd M
LM
Smith
D000478:Alkylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D050814:Octamer Transcription Factor-3 / Q000032:analysis / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D010446:Peptide Fragments; D014357:Trypsin