MSACL 2016 EU Abstract

iST: Sample Preparation for High Throughput Clinical Proteomics

Garwin Pichler (Presenter)
PreOmics GmbH

Authorship: Dr. Garwin Pichler (1), Dr. Nils A. Kulak (1), Dr. Matthias Mann (2)
(1) PreOmics GmbH, (2) Max Planck Institute of Biochemistry

Short Abstract

Sample preparation workflows are a crucial part of routine mass spectrometry (MS) based proteomics measurements. Complex workflows, extensive sample fractionation and proteolytic digestion are highly time consuming and restrict the overall technical reproducibility limiting the overall applicability of MS-based proteomics for clinical applications. The accuracy and robustness of the MS platform is also strongly affected by sample quality reasoning for high quality proteomic samples. Here we present the straightforward, fast, sensitive and robust in-StageTip (iST) method for streamlined sample processing of complete proteomes.

Long Abstract

Introduction

Sample preparation workflows are a crucial part of routine mass spectrometry (MS) based proteomics measurements. Complex workflows, extensive sample fractionation and proteolytic digestion are highly time consuming and restrict the overall technical reproducibility limiting the overall applicability of MS-based proteomics for clinical applications. The accuracy and robustness of the MS platform is also strongly affected by sample quality reasoning for high quality proteomic samples. Here we present the straightforward and robust in-StageTip (iST) method for streamlined sample processing of complete proteomes.

Methods

The iST method is a 3-step procedure performed in a single, enclosed volume which thereby circumvents the likelihood of contamination and sample loss. Due to the straightforward nature, the method can readily be performed in a 96-well format on liquid handling robotic system. The method is highly compatible with established and novel StageTip based pre-fractionation methods and thereby allows in-depth analysis of complex proteomic samples.

Preliminary Data

Applying the procedure to the well-studied cancer cell line HeLa allowed us to estimate protein copy-numbers of 9,667 proteins. The results demonstrated excellent reproducibility (R2 = 0.97) in quadruplicates measurements reflecting the overall strength of the method. In addition, we applied this sample-preparation workflow to process urine samples. We collected urine samples from 6 healthy male donors on 3 consecutive days. We identified a total of 3284 proteins in urine and calculated the label-free quantification values of 2200 proteins in average per sample, with MS-signals spanning 6 orders of magnitude.

Novel Aspect

The in-StageTip method opens up opportunities for high-throughput clinical applications enabling exceptional sample quality at low cost and effort.


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