MSACL 2019 EU Abstract

Semi-Automated Positive Pressure SPE for Proteomics

Stefan Loroch (1), Albert Sickmann (1), Christian Scherling (2) and Shareef Jarvi Antar (2)
(1) Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V., Protein Dynamics, 44227 Dortmund (2) Tecan SP Europe – LC-MS Application Lab, 22335 Hamburg

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Christian Scherling (Presenter) Tecan

Presenter Bio: Christian Scherling works for Tecan as a mass spectrometry automation specialist focusing on MS sample preparation applications for pharma, clinical diagnostics, metabolomics, proteomics, lipidomics and toxicology. His PhD involved metabolomic and proteomic studies, and he has held postdoc positions in metabolomics and systems biology in core facilities. He has extensive experience in automated workflows, consulting with labs around Europe.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

Introduction
Post-translational protein modifications (PTMs) mediate a wide variety of processes in biological systems such as signal transduction, cell cycle regulation or enzymatic activity and abberant PTM have been connected to numerous pathologies. Mass spectrometry-based PTM analysis usually employs tailored analyte enrichment in conjunction with labor-intensive solid-phase extraction (SPE) procedures for initial and final sample clean-up. The positive pressure SPE (ppSPE) workstation allows for semi-automated SPE resulting in significant reduction of lab-time in comparison to manual SPE using spin tips (3–4 hours instead of 2 days for 100 samples). Here we demonstrate how to exploit the workstation in conjunction with ERLIC-HPLC for highly sensitive detection of protein phosphorlyation events in proteomic samples. This workflow includes (I) SPE of samples after digest (RP - Reversed Phase), (II) enrichment and fractionation of phosphopeptides (HPLC-based ERLIC = Electrostatic Repulsion-Hydrophilic Interaction Chromatography) and (III) SPE of fractions for subsequent nanoLC-MS/MS.

Results
In the first experiment, positive pressure SPE with Cerex WWP2 (C18) columns was systemtically compared to manual SPE using SPEC (C18). Both methods showed excellent recovery rates for desalting HeLa digest in the range of 5–500 μg. Below 5 μg both recovery rates were insufficient.In a second experiment, positive pressure SPE with Oasis HLB μElution plates (Waters) performed excellent for recovery of low abundant p-peptides (in comparison to the manual extraction via spin tips). Notably, the automated buffer exchange via the dispensing function of the positive pressure workstation and the transfer to 96 well plate format critically reduced the sample handling time (3–4 h instead of 2 days for 100 samples).

Conclusions & Discussion
In this work we demonstrate the entire semi-automated workflow of proteomics sample prep from protein precipitation, digestion to buffer exchange and clean up experiments in a 96 well plate format. Reproducible protein loading concentrations range from 30 µg to 200 µg for protein digestion in membrane filter, for desalting of tryptic digested HeLa cells using protein loading concentrations from 5 μg to 500 μg were achieved. Recovery rates and signal intensities of peptides and phosphopeptides are equal compare to manual workflow, however the loss of sample during processing was reduced.