Abstract
Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.
Highlights
Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays
parallel reaction monitoring (PRM) methods have the potential of high selectivity by increasing the resolution and the capability to distinguish between target ions and matrix, making it superior compared to conventional Selected Reaction Monitoring (SRM) techniques
Using an in-silico spectral library, pH8 reversed phase sub-fractionation and a PRM screen on selected targets, we identified the peptides of interest central to the key unfolded protein response (UPR) proteins (Supplementary Fig. S1)
Summary
Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. Using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. We highlight the critical instrument parameters of a quadrupole-Orbitrap mass spectrometer needed to increase the sensitivity by at least one order of magnitude allowing the quantification of TFs and TMR down to the low attomol range We applied this workflow to assess the dynamics and copy numbers of the major signaling proteins of the UPR before and after its induction in the glioblastoma cell line LN-308
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