Automating Complex, Multistep Processes on a Single Robotic Platform to Generate Reproducible Phosphoproteomic Data

Abstract

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.

Green leaf volatiles co-opt proteins involved in molecular pattern signaling in plant cells

“…Using an automated workflow 51 and the PolyTi resin for phosphopeptide enrichment, 51–53 we identified ~ 1700 phosphoproteins at medium to high confidence. The average specificity of phosphopeptide enrichment was ≥ 79% (Fig.…”

“…Ethanol (15 µL) was added to the control group, and a treatment with 10 nM systemin served as a reference. Treated SP cells were lysed using the method described by Mullis et al 51 . Briefly, 1 mL of RIPA buffer (Thermo Scientific Pierce, Rockford, IL) containing 1× Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific Pierce) was added to each sample.…”

“…Then, samples were desalted using 5 mg RP 300 μL IMCStips (IMCS, Irmo, SC), followed by phosphopeptide enrichment using 10 μg PolyTi 300 μL IMCStips (IMCS, Irmo, SC). The automated peptide desalting and phosphopeptide enrichment was performed on a Hamilton STAR system following the previously published workflow 51 . The eluted solutions were lyophilized and stored at −80 °C until further analysis.…”

Phosphopeptide enrichment for phosphoproteomic analysis - A tutorial and review of novel materials

“…Developments of new supporting formats are 731 necessary for enabling high-throughput approaches, due to the multiple manual 732 handling steps required for these protocols. A few exciting new examples of these support formats include: the lab-in-syringe mode of Ti 4+ @PDA@GA (graphene aerogel, GA) [53]; the TiO 2 nanoparticle packed channel array glass microchip [71]; instrument-free TiO 2 -modified filter paper-based analytical device [72]; "green 736 synthesised" Ti 4+ -IMAC carbonaceous spheres using glucose, vinylphosphonic acid and water solvent [73] automated p-peptide enrichment and desalting tip set up [74]. All of these methods are time and cost effective, compared with the commonly used batch mode or micro-column format packed materials.…”

Parallel Sample Processing using Dispersive INtip Micro-purification on Programmable Multichannel Pipettes

“…Applications of dSPE are expanding; recent uses include detection of contaminating analytes in pork [7] and wine [12] and the extraction of urine catecholamines [13]. Recently, our lab has reported a fully automated method for the identification of phosphopeptides from cell lysate using INtip technology [14]. Much of the previous work with dSPE tips employed organic solvents to aid subsequent separations [15,16].…”