FLEXIQuant: A Novel Tool for the Absolute Quantification of Proteins, and the Simultaneous Identification and Quantification of Potentially Modified Peptides

Abstract

Measurements of protein abundance and quantitative assessment of multiple post-translational modifications (PTMs) within a single protein are increasingly called for to understand the control of protein activity, particularly in metazoan cells. General methods of wide applicability and precision/accuracy for quantitative estimation of protein post-translational regulation are lacking. Protein mass spectrometry has evolved from a high throughput qualitative technique to a potentially general quantitative tool but there are still serious limitations in dynamic range and coverage. To address some of these limitations we introduce a novel MS-based quantitative strategy, FLEXIQuant, (Full-Length Expressed Stable Isotope-labeled Proteins for Quantification), which can track changes in relative peptide abundances as a function of PTM, and determine absolute quantity of a protein from its lysate. We examined two subunits of the anaphase-promoting complex, CDC27 and APC5, as a test of our ability to monitor quantitatively, the PTM status of several peptides over time. We find evidence of differential regulation at different sites, a phenomenon we believe will be very widespread. FLEXIQuant proved itself to be capable of serving as a general quantitative tool.

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics

“…The use of isotope-labeled full-length proteins offers clear advantages over other recently developed quantification approaches (Supplementary Table 1). Both synthetic-and recombinant-based approaches can introduce single reference [16][17][18][19] or solubility (for example, albuminbinding protein 15 or glutathione S-transferase 14 ) tags linked to parent proteins. This enables the stoichiometric quantification of peptide standards released upon protein digestion, and these standards are then used to quantify the endogenous protein in a complex sample.…”

“…In-gel separation followed by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the tagged TF-containing fraction enabled us to generate a collection of experimentally detected TF-specific tryptic peptides for the ten proteins (Supplementary Data 1 and Online Methods). 18,19,25 into the gel-based workflow described above for transition selection. This strategy relies on the simplification of full-length protein quantification using reporter peptide tags 18,19 and on the increased detection sensitivity provided by SRM-based mass spectrometry measurements.…”

“…18,19,25 into the gel-based workflow described above for transition selection. This strategy relies on the simplification of full-length protein quantification using reporter peptide tags 18,19 and on the increased detection sensitivity provided by SRM-based mass spectrometry measurements. For this purpose, we aimed to add an in-frame proteotypic referencepeptide tag at the N terminus of the protein constructs that should serve as surrogate during the quantification step.…”

“…For this purpose, we aimed to add an in-frame proteotypic referencepeptide tag at the N terminus of the protein constructs that should serve as surrogate during the quantification step. After having considered several possibilities 18,19 (Online Methods), we found that the SH-quant peptide 19 offers optimal solubility and stability properties (Supplementary Fig. 3).…”

Discordance between protein and transcript levels detected by selected reaction monitoring

“…Furthermore, SRM analysis allows the quantification of absolute abundances of posttranslational modifications such as phosphorylation states. [25][26][27] Although less common in the field of plant science, this method also has the potential to be used for other purposes, such as the quantification of stoichiometric changes in protein complexes dependent on environmental stimuli, or the quantification of developmental stages.…”