Niles Richard, Witkowska H Ewa, Allen Simon, Hall Steven C, Fisher Susan J, Hardt Markus
Department of Cell and Tissue Biology, University of CaliforniaSan Francisco, San Francisco, California 94143, USA.
Anal Chem. 2009 Apr 1;81(7):2804-9. doi: 10.1021/ac802484d.
In enzymatic (18)O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as method for introducing (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable-isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after approximately 11 days. Reaction rates were residue- and sequence-specific. Due to its slow nature, the acid-catalyzed back exchange is expected to minimally interfere with enzymatic (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics.
在定量蛋白质组学的酶促（18）O标记策略中，低pH下羧基氧的交换是一种常见的、不期望发生的副反应。我们探讨了酸催化的反向交换是否会干扰定量分析，以及该反应本身是否可作为将（18）O标记引入肽段的方法。将几种合成肽溶解于含有50%（v/v）H₂（18）O的稀酸中，并在室温下孵育。在3周的时间内定期取出等分试样，通过串联质谱（MS/MS）进行分析。通过允许多次稳定同位素掺入的线性回归分析来确定（18）O掺入率。在低pH下，肽段将其羧基氧原子与水性溶剂进行交换。同位素模式逐渐向更高质量转移，直到大约11天后达到平衡时的预期二项分布。反应速率具有残基和序列特异性。由于其反应缓慢，如果储存和分析条件能将低pH暴露时间降至最低，预计酸催化的反向交换对酶促（18）O标记研究的干扰最小。就其本身而言，酸催化的（18）O标记是一种通用的标记策略，是目前定量蛋白质组学中使用的化学、代谢和酶促同位素标记方案的替代方法。
