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.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…In pilot experiments, we identified the two most suited PTPs for each centrosomal protein to be quantified (Appendix Fig S1), as detailed in Appendix Fig S2, and acquired isotope‐labeled synthetic versions of these peptides (AQUA peptides; Gerber et al , 2003). After using these heavy reference peptides for optimizing collision energies for all selected SRM transitions, the linear dynamic range, reproducibility, and quantification limits of each SRM assay were thoroughly assessed through analyses of dilution series with whole human cell lysates (Bauer et al , 2014). Although wide dynamic ranges were observed for all assays (> 4 orders of magnitude), extract fractionation by Off‐Gel electrophoresis was required to further improve the sensitivity of SRM assays (Picotti et al , 2009) and allow confident detection and quantification of the least abundant target proteins.…”

“…Although wide dynamic ranges were observed for all assays (> 4 orders of magnitude), extract fractionation by Off‐Gel electrophoresis was required to further improve the sensitivity of SRM assays (Picotti et al , 2009) and allow confident detection and quantification of the least abundant target proteins. By comparing transition intensities of “heavy” reference peptides (spiked in known amounts into tryptic digests prior to any fractionation) and their corresponding “light” counterparts derived from endogenous proteins (Appendix Fig S3), the abundance of endogenous proteins in whole‐cell extracts could be accurately determined (Bauer et al , 2014). …”

“…Purified peptides were dried at 45°C under vacuum and resuspended in 200 μl of 10% ACN/90% water (v/v) and subjected to Off‐Gel electrophoresis (OGE) using 24‐cm strips with a pH range from 3 to 10 (3100 OFFGEL Fractionator, Agilent technologies, Santa Clara, CA, USA). The 24 OGE fractions were purified using microspin solid‐phase extraction C18 columns according to the manufacturer's instructions (The Nest Group, Southborough, MA, USA), dried at 45°C under vacuum, resuspended in an aqueous solution containing 5% ACN/0.15% formic acid, and subjected to SRM analysis in a TSQ Vantage mass spectrometer (Thermo Scientific, Waltham, MA, USA) as recently described (Bauer et al , 2014). Cycle time was set to 2 s resulting in a dwell time of 20 ms per transition.…”

“…Cycle time was set to 2 s resulting in a dwell time of 20 ms per transition. The transition lists with optimized collision energies for all peptides were reported previously (Bauer et al , 2014). Ratios between heavy‐labeled AQUA peptides and light endogenous peptides were determined using the Skyline software (MacLean B. et al , Bioinformatics, 2010; version 1.3).…”

“…In particular, selected reaction monitoring (SRM) allows the detection of specific proteins in complex mixtures with high sensitivity and over a broad dynamic range (Picotti et al , 2009; Bauer et al , 2014). In parallel, EGFP‐tagging of endogenous proteins through genetic engineering offers opportunities for quantifying fluorescence signals at specific subcellular locations (e.g.…”