Evaluation of Data-Dependent and -Independent Mass Spectrometric Workflows for Sensitive Quantification of Proteins and Phosphorylation Sites
详细信息 查看全文
- 作者：Manuel Bauer ; Erik Ahrn茅 ; Anna P. Baron ; Timo Glatter ; Luca L. Fava ; Anna Santamaria ; Erich A. Nigg ; Alexander Schmidt
- 刊名：Journal of Proteome Research
- 出版年：2014
- 出版时间：December 5, 2014
- 年：2014
- 卷：13
- 期：12
- 页码：5973-5988
- 全文大小：828K
- ISSN：1535-3907
文摘
In recent years, directed and, particularly, targeted mass spectrometric workflows have gained momentum as alternative techniques to conventional data-dependent acquisition (DDA) LC鈥揗S/MS approaches. By focusing on specific peptide species, these methods allow hypothesis-driven analysis of selected proteins of interest, and they have been shown to be suited to monitor low-abundance proteins within complex mixtures. Despite their growing popularity, no study has systematically evaluated these various MS strategies in terms of quantification, detection, and identification limits when they are applied to complex samples. Here, we systematically compared the performance of conventional DDA, directed, and various targeted MS approaches on two different instruments, namely, a hybrid linear ion trap鈥揙rbitrap and a triple quadrupole instrument. We assessed the limits of identification, quantification, and detection for each method by analyzing a dilution series of 20 unmodified and 10 phosphorylated synthetic heavy-labeled reference peptides, respectively, covering 6 orders of magnitude in peptide concentration with and without a complex human cell digest background. We found that all methods performed similarly in the absence of background proteins; however, when analyzing whole-cell lysates, targeted methods were at least 5鈥?0 times more sensitive than that of the directed or DDA method. In particular, higher stage fragmentation (MS3) of the neutral loss peak using a linear ion trap increased the dynamic quantification range of some phosphopeptides up to 100-fold. We illustrate the power of this targeted MS3 approach for phosphopeptide monitoring by successfully quantifying nine phosphorylation sites of the kinetochore and spindle assembly checkpoint component Mad1 over different cell cycle states from nonenriched pull-down samples.