title: Evaluation of data-dependent and -independent mass spectrometric workflows for sensitive quantification of proteins and phosphorylation sites.
abstract: In recent years, directed and, particularly, targeted mass spectrometric workflows have gained momentum as alternative techniques to conventional data-dependent acquisition (DDA) LC-MS/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--Orbitrap 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 5-10 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.
authors.affiliation: Biozentrum, University of Basel , Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland.
authors.forename: Manuel
authors.initials: M
authors.lastname: Bauer
authors.forename: Erik
authors.initials: E
authors.lastname: Ahrné
authors.forename: Anna P
authors.initials: AP
authors.lastname: Baron
authors.forename: Timo
authors.initials: T
authors.lastname: Glatter
authors.forename: Luca L
authors.initials: LL
authors.lastname: Fava
authors.forename: Anna
authors.initials: A
authors.lastname: Santamaria
authors.forename: Erich A
authors.initials: EA
authors.lastname: Nigg
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmidt
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D018797:Cell Cycle Proteins; D002457:Cell Extracts; C501384:MAD1L1 protein, human; D009687:Nuclear Proteins; D010748:Phosphopeptides
keywords: MAD1; Targeted mass spectrometry; cell cycle; data-dependent acquisition; directed mass spectrometry; limit of detection; peptide quantification; phosphorylation