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Updated: Apr 21, 2026
Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry
Published on: April 4, 2018
Evaluating nonpolar surface area and liquid chromatography/mass spectrometry response: an application for site
Shan M Randall1, Irina Koryakina, Gavin J Williams
1W.M. Keck Fourier Transform Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA; Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Rationale:
Site occupancy measurements using liquid chromatography/mass spectrometry (LC/MS) are reported throughout the literature. However, site occupancy quantification suffers from ionization bias between modified and unmodified peptides containing the active site. In this study, we explore the MS signal as a function of nonpolar surface area (NPSA) in order to better understand this bias in electrospray response. The correlation between hydrophobicity and LC/MS response was evaluated and applied to study enzyme intermediates in polyketide synthases.
Methods:
Site occupancy methods were developed to study acyltransferase activity. To further evaluate these methods, several standard peptides containing one cysteine residue were modified with alkylation reagents of increasing hydrophobicity to study the MS signal as a function of NPSA.
Results:
A consistent trend in MS response was observed which is dependent on the NPSA of the analyte. An optimal NPSA zone was observed for the peptides studied.
Conclusions:
Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.
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