Comparative Assessment of Quantification Methods for Tumor Tissue Phosphoproteomics

Abstract

With increasing sensitivity and accuracy in mass spectrometry, the tumor phosphoproteome is getting into reach. However, the selection of quantitation techniques best-suited to the biomedical question and diagnostic requirements remains a trial and error decision as no study has directly compared their performance for tumor tissue phosphoproteomics. We compared label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling by amino acids in cell culture), and tandem mass tag (TMT) isobaric tandem mass tags technology for quantitative phosphosite profiling in tumor tissue. Compared to the classic SILAC method, spike-in-SILAC is not limited to cell culture analysis, making it suitable for quantitative analysis of tumor tissue samples. TMT offered the lowest accuracy and the highest precision and robustness toward different phosphosite abundances and matrices. Spike-in-SILAC offered the best compromise between these features but suffered from a low phosphosite coverage. LFQ offered the lowest precision but the highest number of identifications. Both spike-in-SILAC and LFQ presented susceptibility to matrix effects. Match between run (MBR)-based analysis enhanced the phosphosite coverage across technical replicates in LFQ and spike-in-SILAC but further reduced the precision and robustness of quantification. The choice of quantitative methodology is critical for both study design such as sample size in sample groups and quantified phosphosites and comparison of published cancer phosphoproteomes. Using ovarian cancer tissue as an example, our study builds a resource for the design and analysis of quantitative phosphoproteomic studies in cancer research and diagnostics.

Rapid and High-Sensitive Phosphoproteomics Elucidated the Spatial Dynamics of the Mouse Brain

“…Furtherly, to assess the effect of the newly developed miniPhos method, we compared it with a commercial kit (High-Select Fe-NTA Phosphopeptide Enrichment Kit) that is widely used for phosphopeptide enrichment, especially in clinical samples. − To this end, we conducted an experiment in which 100 μg of the HeLa cell proteins was processed by miniPhos (TiO 2 for enrichment) or the Fe-NTA Kit in parallel. A significantly higher number of phosphopeptides and phosphosites were found using the miniPhos method (adjusted P < 0.01), with six orders of magnitude in phosphopeptide intensities (Figure A,B).…”

“…The current use of powerful mass spectrometry (MS) in single-cell proteomics has paved the way for deeper and more sensitive investigations. , However, it remains challenging for phosphoproteomic analysis of low-input samples, as phosphoproteomics typically requires 100-fold more starting material than proteome profiling. Phosphopeptides comprise less than 2–3% of peptides in a typical tryptic digest, which limits the phosphorylation analysis for trace samples. , Especially in clinical studies, the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific, MO, USA) was widely used, for which at least 500 μg of peptides was required. − However, this limits the application on some rare and precious samples such as fine-needle aspiration biopsies that a tumor area of <5 mm 2 (10 μm thick) only yields 70 μg of protein . Numerous strategies have been made for effective and selective enrichment of phosphopeptides and even for mini-scale samples. − One high-throughput workflow called easyPhos quantified over 15,000 phosphopeptides with 200 μg of protein starting material .…”

Advancements in Oncoproteomics Technologies: Treading toward Translation into Clinical Practice

“…It can be applied to intact proteins and could enable robust, multiplexed quantitation for top-down experiments [ 161 ]. The advancements in SILAC have enhanced our understanding of cancer biology and serve as a tool for biomarker discovery [ 169 , 170 , 171 , 172 , 173 , 174 , 175 ].…”