Assessment of SRM, MRM³, and DIA for the targeted analysis of phosphorylation dynamics in non‐small cell lung cancer

Abstract

Hypothesis-driven MS-based targeted proteomics has gained great popularity in a relatively short timespan. Next to the widely established selected reaction monitoring (SRM) workflow, data-independent acquisition (DIA), also referred to as sequential window acquisition of all theoretical spectra (SWATH) was introduced as a high-throughput targeted proteomics method. DIA facilitates increased proteome coverage, however, does not yet reach the sensitivity obtained with SRM. Therefore, a well-informed method selection is crucial for designing a successful targeted proteomics experiment. This is especially the case when targeting less conventional peptides such as those that contain PTMs, as these peptides do not always adhere to the optimal fragmentation considerations for targeted assays. Here, we provide insight into the performance of DIA, SRM, and MRM cubed (MRM(3) ) in the analysis of phosphorylation dynamics throughout the phosphoinositide 3-kinase mechanistic target of rapamycin (PI3K-mTOR) and mitogen-activated protein kinase (MAPK) signaling network. We observe indeed that DIA is less sensitive when compared to SRM, however demonstrates increased flexibility, by postanalysis selection of alternative phosphopeptide precursors. Additionally, we demonstrate the added benefit of MRM(3) , allowing the quantification of two poorly accessible phosphosites. In total, targeted proteomics enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites, gaining a so far unachieved in-depth view mTOR signaling events linked to tyrosine kinase inhibitor resistance in non-small cell lung cancer.

Comparison of targeted proteomics approaches for detecting and quantifying proteins derived from human cancer tissues

“…Schmiddlin et al. quantified PI3K‐mTOR/MAPK involved phosphopeptides, concluding that SRM was more sensitive than SWATH while quantitative data had a good correlation ( R 2 = 0.82) , which is in principal agreement with our data. In a contrary to all above studies, the analysis of microsomal membrane proteins by Nakamura et al led to conclusion that SWATH was even more sensitive than SRM and p‐SRM/PRM .…”

“…However, there is a trade‐off between the sensitivity and level of multiplexing for SRM, p‐SRM/PRM, and SWATH . Although there are some previous studies comparing the sensitivity between SRM, p‐SRM/PRM, and SWATH in different samples such as yeast digest , plasma glycopeptides , lung cancer cell phosphopeptides , pooled human microsomes , urine samples , and human plasma , there is no report directly comparing these three methods for analyzing the clinically important cancer tissue extracts to date. Here we analyzed the model breast cancer samples by using SRM, p‐SRM, and SWATH to compare their key analytical parameters: signal‐to‐noise (S/N), noise, coefficient of variance (CV), limit of detection and quantitation (LOD, LOQ), and biological differences between samples (fold change, FC).…”

Quantitative proteomics in lung cancer

“…An alternative method using targeted proteomics by SRM and MRM with sensitive detection enable us to identify specific PTM and give the absolute copy number of proteins in a single cell. Recent study of phosphorylation dynamics in non-small cell lung cancer by targeted proteomics including SRM, MRM, and DIA enabled the quantification of 42 PI3K-mTOR and MAPK phosphosites and provides valuable conclusion on each assessment [74]. Quantitative proteomics provides the information for synthetic biologists to engineer or rewire the key pathways, furthermore to offers the best therapeutic strategy for lung cancer.…”

Clinical applications of quantitative proteomics using targeted and untargeted data-independent acquisition techniques

“…Many diseases are known to cause changes in their dynamic PTM signaling profile, specifically in cancer affecting phosphorylation patterns which provides interesting experimental designs when using uDIA for their analysis [76,78,79]. Other studies have focused on histone modifications, such as in an elegant PRM study published by Jaffe and colleagues describing the targeted quantification histone PTMs [75], but also recent uDIA studies [80,81].…”