Randall Shan M, Koryakina Irina, Williams Gavin J, Muddiman David C
W.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.
Rapid Commun Mass Spectrom. 2014 Dec 15;28(23):2511-22. doi: 10.1002/rcm.7051.
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.
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.
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.
Nonpolar surface area can be used as metric to determine relative LC/MS response for peptides and evaluate site occupancy measurements.
文献中报道了使用液相色谱/质谱（LC/MS）进行位点占有率测量的方法。然而，位点占有率定量存在含活性位点的修饰肽和未修饰肽之间的电离偏差。在本研究中，我们探索了作为非极性表面积（NPSA）函数的质谱信号，以便更好地理解电喷雾响应中的这种偏差。评估了疏水性与LC/MS响应之间的相关性，并将其应用于研究聚酮合酶中的酶中间体。
开发了位点占有率方法来研究酰基转移酶活性。为了进一步评估这些方法，用疏水性增加的烷基化试剂修饰了几个含有一个半胱氨酸残基的标准肽，以研究作为NPSA函数的质谱信号。
观察到质谱响应的一致趋势，该趋势取决于分析物的NPSA。在所研究的肽中观察到一个最佳的NPSA区域。
非极性表面积可作为一种度量标准，用于确定肽的相对LC/MS响应并评估位点占有率测量。
