Optimized Suspension Trapping Method for Phosphoproteomics Sample Preparation

Abstract

A successful mass spectrometry-based phosphoproteomics analysis relies on effective sample preparation strategies. Suspension trapping (S-Trap) is a novel, rapid, and universal method of sample preparation that is increasingly applied in bottom-up proteomics studies. However, the performance of the S-Trap protocol for phosphoproteomics studies is unclear. In the existing S-Trap protocol, the addition of phosphoric acid (PA) and methanol buffer creates a fine protein suspension to capture proteins on a filter and is a critical step for subsequent protein digestion. Herein, we demonstrate that this addition of PA is detrimental to downstream phosphopeptide enrichment, rendering the standard S-Trap protocol suboptimal for phosphoproteomics. In this study, the performance of the S-Trap digestion for proteomics and phosphoproteomics is systematically evaluated in largescale and small-scale samples. The results of this comparative analysis show that an optimized S-Trap approach, where trifluoroacetic acid is substituted for PA, is a simple and effective method to prepare samples for phosphoproteomics. Our optimized S-Trap protocol is applied to extracellular vesicles to demonstrate superior sample preparation workflow for low-abundance, membrane-rich samples.

In-Depth Endogenous Phosphopeptidomics of Serum with Zirconium(IV)-Grafted Mesoporous Silica Enrichment

“…Moreover, our analysis results are comparable to or even better than a number of recent phosphoproteomics studies in which equal or similar amounts of HeLa cell digests were used, and single-shot mass spec. analyses were performed. , ,, − Given that phosphorylation levels in serum samples are much lower (in contrast to complex samples such as whole cell lysates and tissue samples), we think it is decent enough to perform single-shot analyses and comparisons (rather than doing extensive fractionation for DDA-based phosphoproteomics or DIA-based phosphoproteomics). − While many phosphosites were identified by different approaches, still a distinct set of phosphosites could be found by each method (Figure S3B). As shown in Figure S3C, a total of 8314 phosphopeptides were identified after Zr-IMAC@MCM-48 enrichment, comprising 5680 singly (68.4%), 2058 doubly (24.8%), and 572 triply phosphorylated peptides (6.9%), which is similar to the results obtained with Fe-NTA.…”

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap)

“…Chemoproteomics is increasingly performed in large scale, aided by techniques such as single-pot, solid-phase-enhanced sample-preparation (SP3), clickable probes and multi-/hyperplexing. , Similarly, compared to ABE and Acyl-RAC, Acyl-Trap helps overcome the “throughput barriers” associated with traditional protein precipitation and recovery. While suspension trapping has been used in series with phosphopeptide enrichment, and for on-trap isotopic Cys labeling, our demonstration of its compatibility with thioester cleavage and in situ Cys capture, at the protein and peptide level, serves as a proof-of-principle for the utility of suspension trapping in covalent PTM-enrichment. Given the wide range of commercially available organosilanes, a future toolbox of “chemically tailored” suspension traps may facilitate a range of targeted enrichments and allow PTMs (including S -acylation) to become a component of standardized (chemo)­proteomic workflows for drug discovery and large scale ‘omics.…”

FAS Inhibited Proteomics and Phosphoproteomics Profiling of Colorectal Cancer Spheroids Shows Activation of Ferroptotic Death Mechanism

“…Reduced and denatured proteins were alkylated with 40 mM IAA for 30 min in the dark. Alkylated proteins were acidified to 0.9% TFA according to a previous report . Acidified proteins were then diluted with 90% methanol and 100 mM TEAB and spun onto the S-Trap mini column via centrifugation at 4,000 rpm for 1 min.…”