Mass spectrometry-based phosphoproteomics holds promise for advancing drug treatment and disease diagnosis; however, its clinical translation has thus far been limited. This is in part due to an unstandardized and segmented sample preparation process that involves cell lysis, protein digestion, peptide desalting, and phosphopeptide enrichment. Automating this entire sample preparation process will be key in facilitating standardization and clinical translation of phosphoproteomics. While peptide desalting and phosphopeptide enrichment steps have been individually automated, integrating these two extractions and, further, the entire process requires more advanced robotic platforms as well as automation-friendly extraction tools. Here we describe a fully automated peptide desalting and phosphopeptide enrichment method using IMCStips on a Hamilton STAR. Using our established automated method, we identified more than 10,000 phosphopeptides from 200 µg of HCT116 cell lysate without fractionation with >85% phosphopeptide specificities. Compared with titania-based Spin Tip products, the automated IMCStips-based method gave 50% higher phosphopeptide identifications. The method reproducibility was further assessed using multiple reaction monitoring (MRM) to show >50% phosphopeptide recoveries after the automated phosphopeptide extraction with coefficients of variation (CVs) of <20% over a 3-week period. The established automated method is a step toward standardization of the sample preparation of phosphopeptide samples and could be further expanded upon to create a fully automated “cells to phosphopeptides” method.
A dynamic intramolecular charge-transfer (CT) complex was designed that displayed reversible colour changes in the solid-state when treated with different organic solvents. The origins of the dichromatism were shown to be due to solvent-inclusion, which induced changes in the relative orientations of the donor pyrene and acceptor naphthalenediimide units.
The green leaf volatiles (GLVs) Z-3-hexen-1-ol and Z-3-hexenyl acetate are airborne infochemicals released from damaged plant tissues that prime defenses against herbivores and pathogens in receiver plants. They are conceptually similar to well-known damage-associated molecular patterns (DAMPs), but little is known about their mechanism of action. Using tomato cell cultures, we found that rapid responses to the two GLVs and the polypeptide DAMP systemin showed a significant overlap but also GLV-specific patterns. Within five minutes, GLVs induced changes in MAPK activity and proton-fluxes as well as rapid and massive changes in the phosphorylation status of proteins. Many of these proteins are involved in reprogramming the proteome from cellular homeostasis to stress and include pattern recognition receptors, a receptor-like cytoplasmic kinase, MAPK cascade components, calcium signaling proteins, and transcriptional regulators, all of which are also components of DAMP signaling pathways. This phosphoproteome may represent an early priming state that enables plants to respond forcefully to a subsequent stress signal.
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