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European Biopharmaceutical Review

Perfecting Progress

Mass spectrometry (MS)-based proteomics has evolved to be a powerful technique generating large but complex protein datasets. For a long time, however, this field lacked guidelines and standardised methods to assess the quality of the information produced (1-3). Standardisation and quality assessment of an MS-based proteomics workflow is not straightforward – it is complicated by the diverse array of mass spectrometers used; the many different types of experiments generating proteomics samples; and the multiple ways of analysing the data. Recently, the development of computational tools covering different aspects of an MS-based proteomics experiment emerged to quality control (QC) MS data. These tools either monitor quality at the instrument, spectral or identification level (4). However, at the level of sample preparation, standardised QC steps are still lacking, and experimental design is a key component in generating highly qualitative data.

LC-MS/MS

A first tendency towards standardisation in proteomics sample preparation workflows is a purification step of samples designated for liquid chromatography-tandem MS (LC-MS/MS) analysis using solid-phase extraction (5). While this increases sample purity, quantification at the peptide level is non-existent – but it is generally accepted that injecting a non-optimal peptide amount is detrimental to the quality and read depth of proteomics data. Accurate peptide quantification is, furthermore, beneficial for labelfree quantification (LFQ) strategies as equal amounts of comparative samples can be loaded (6).

In a standard bottom-up proteomics experiment, the total amount of protein present in a sample is typically determined following protein extraction, and a second time before protein digestion. Whether or not additional modifications or enrichment steps at the peptide level are performed, a QC step before the actual LC-MS/MS analysis is generally not carried out. Moreover, in several cases, even quantification at the protein level is difficult or close to impossible due to inadequate protein amounts – for example, upon affinity purification of protein complexes. Injecting inadequate amounts of peptides, however, will have an impact on the number of peptides and proteins identified and may lead to additional LC-MS/MS analysis of the same sample.

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Kim Plasman is Application Scientist at Trinean and holds a PhD in Biomedical Sciences from Vlaams Instituut voor Biotechnologie (VIB) at Ghent University, Belgium. Within the Applications team, Kim is responsible for the design and development of customer-specific applications independent of sample source.

Kris Gevaert holds a PhD in Biotechnology from Ghent University. He is currently a Full Professor at Ghent University and Associate Department Director of the VIB-UGent Center for Medical Biotechnology. His group has published more than 280 papers and several book chapters on the development and applications of proteomics techniques in numerous areas of biomedical and life sciences research.

Francis Impens is a Biomedical Researcher and Head of the VIB Proteomics Core hosted at Ghent University. His team provides proteomics services to the academic and industrial research community and further focuses on the development of novel MSbased approaches to study host-pathogen interactions on the protein and post‐translational levels. 

Tony Montoye is Head of Marketing and Applications at Trinean and holds a PhD in Biomedical Sciences from VIB at Ghent University. Joining Trinean from the startup phase, Tony has been involved in R&D, commercial and business aspects – growing the company from a university spin-off to its mature state today. His current focus lies in driving marketing and product strategy, expanding the customer base and leading a team of motivated application developers. 
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Kim Plasman
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Kris Gevaert
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Francis Impens
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Tony Montoye
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