Mirza U A, Chait B T
Rockefeller University, New York, New York 10021.
Anal Chem. 1994 Sep 15;66(18):2898-904. doi: 10.1021/ac00090a017.
Positive ion electrospray ionization mass spectra of polypeptides are usually obtained from solutions that are acidified and therefore contain relatively high concentrations of anions. The present study describes an investigation of the effects of these ubiquitous anions on the positive ion electrospray ionization mass spectra of peptides and proteins. Certain anionic species in the spray solutions were observed to cause a marked decrease in the net average charge of peptide and protein ions in the mass spectra compared to the average charge measured in the absence of these anions. This charge neutralization effect was found to depend solely on the nature of the anionic species and was independent of the source of the anion (acid or salt), with the propensity for neutralization following the order: CCl3COO- > CF3COO- > CH3COO- approximately Cl-. A mechanism for the observed charge reduction effect is proposed that involves two steps. The first step occurs in solution, where an anion pairs with a positively charged basic group on the peptide. The second step occurs during the process of desolvation or in the gas phase, where the ion pair dissociates to yield the neutral acid and the peptide with reduced charge state. The different propensities for charge neutralization of the different anionic species is presumed to reflect the avidity of the anion-peptide interaction. These findings demonstrate that any attempt to correlate the distribution of charge states observed on proteins in the gas phase (by positive ion electrospray ionization mass spectrometry) with the net charge residing on the protein in solution will require that the described anion effect be taken into account. In addition, it appears that some control over the distribution of charge states on peptides and protein ions can be exercised by an appropriate choice of anion in the electrospray solution.
多肽的正离子电喷雾电离质谱通常从酸化的溶液中获得，因此这些溶液含有相对较高浓度的阴离子。本研究描述了对这些普遍存在的阴离子对肽和蛋白质的正离子电喷雾电离质谱影响的调查。与在不存在这些阴离子的情况下测得的平均电荷相比，喷雾溶液中的某些阴离子种类会导致质谱中肽和蛋白质离子的净平均电荷显著降低。发现这种电荷中和效应仅取决于阴离子种类的性质，并且与阴离子的来源（酸或盐）无关，中和倾向遵循以下顺序：CCl3COO- > CF3COO- > CH3COO- ≈ Cl-。提出了一种观察到的电荷减少效应的机制，该机制涉及两个步骤。第一步发生在溶液中，阴离子与肽上带正电荷的碱性基团配对。第二步发生在去溶剂化过程中或气相中，离子对解离产生中性酸和电荷状态降低的肽。推测不同阴离子种类的电荷中和倾向反映了阴离子 - 肽相互作用的亲和力。这些发现表明，任何试图将气相中蛋白质上观察到的电荷状态分布（通过正离子电喷雾电离质谱法）与溶液中蛋白质上的净电荷相关联的尝试都需要考虑上述阴离子效应。此外，似乎通过在电喷雾溶液中适当选择阴离子，可以对肽和蛋白质离子上的电荷状态分布进行一些控制。
