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Home > Science > Mass spectrometry

Introduction

Proteins carry out a very large number of functions in cells from providing scaffolds and basic structures to catalyzing chemical reactions. Mass spectrometry (MS) is one of the best suited tools to study proteins as it is fast and sensitive and can be used to identify proteins and various modifications (either biological (post-translational modifications) or synthetic (such as chemical cross links)) and quantify both relative between various conditions or absolutely by using references.

One common way of using an MS in biology is to lyze cells and digest all proteins into peptides using a protease such as trypsin. These peptides are then separated using liquid chromatography (LC) and the LC columns is directly interfaced with an MS using an ionization source called Electrospray Ionization (ESI). The ESI creates droplets and the protonated peptides gets ripped out of these droplets using a strong electric field. The charge/mass ratio (m/z) is measured for all peptide ions and one or more ion can be selected for fragmentation. Fragmentation can be accomplished by colliding the peptide ion with an inert gas which results in the breakage of peptide bonds. Since one or more of the resulting fragments are still charged, these fragments can be accelerated and the m/z for these fragments can be measured. The resulting spectra, sometimes referred to as an MS/MS or MS2 spectra can be used to determine the sequence of the peptide that was fragmented. This information in turn can be used to determine which protein was digested. The MS is fast and can generate thousands of MS/MS spectra in an hour.

XL-MS - cross-linking mass spectrometry

Proteins rarely work in isolation but assemble into small and large protein complexes. It is becoming more obvious that knowing the structure of protein complexes is necessary to understand the function and role of individual proteins. As all structure is lost during sample preparation, it is common to modify the sample before sample preparation in such a way that the protein complex structure information can be inferred. One way of doing this is to use chemical crosslinks that covalently attached two amino acids of, ideally, different protein chains. The proteins are then digested and the crosslinked peptides are measured. My colleagues at the ETH have greatly improved this technology in recent years and this allows us to quickly generate distance constraints. These constraints are then used to guide protein-protein docking simulations.

Reference
Kahraman, Abdullah; Herzog, Franz; Leitner, Alexander; Rosenberger, George; Aebersold, Ruedi; Malmstrom, Lars; Cross-Link Guided Molecular Modeling with ROSETTA. PLoS One (2013), 8: e73411.
Herzog, Franz; Kahraman, Abdullah; Boehringer, Daniel; Mak, Raymond; Bracher, Andreas; Walzthoeni, Thomas; Leitner, Alexander; Beck, Martin; Hartl, Franz-Ulrich; Ban, Nenad; Malmstrom, Lars; Aebersold, Ruedi; Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry. Science (2012), 337: 1348-52.

SRM MS - targeted data acquisition

SRM MS, or Selected Rection Monitoring MS, is an acquisition technique where the user defines exactly which peptides to measure beforehand. The MS ignores any ions not specified and this allows the mass spectrometer to generate less, but more informative data. SRM MS is also more sensitive than any other MS technique.

Reference
Karlsson, Christofer; Malmstrom, Lars; Aebersold, Ruedi; Malmstrom, Johan; Proteome-wide selected reaction monitoring assays for the human pathogen Streptococcus pyogenes. Nat Commun (2012), 3: 1301.
Reker, Daniel; Malmstrom, Lars; Bioinformatic challenges in targeted proteomics. J Proteome Res (2012), 11: 4393-402.
Rost, Hannes; Malmstrom, Lars; Aebersold, Ruedi; A computational tool to detect and avoid redundancy in selected reaction monitoring. Mol Cell Proteomics (2012), 11: 540-9.
Malmstrom, Lars; Malmstrom, Johan; Selevsek, Nathalie; Rosenberger, George; Aebersold, Ruedi; Automated workflow for large-scale Selected Reaction Monitoring experiments. J Proteome Res (2012), 11: 1644-53.
Malmstrom, Johan; Karlsson, Christofer; Nordenfelt, Pontus; Ossola, Reto; Weisser, Hendrik; Quandt, Andreas; Hansson, Karin; Aebersold, Ruedi; Malmstrom, Lars; Bjorck, Lars; Streptococcus pyogenes in human plasma: adaptive mechanisms analyzed by mass spectrometry based proteomics. J Biol Chem (2011), 287: 1415-25.

Label-free quantification

Measuring difference in protein abundance between samples can be informative in multiple ways. In label-free quantification, the signal in the MS1 spectra are integrated and serves as a proxy for abundance. For example, differences in protein expression between a healthy and diseased state can provide clues about both what is causing the disease and how the cells respond to various stimuli. My research attempts to find ways to improve so-called label-free quantification technology in which no chemical modification (labeling) has been performed on the samples. The advantages of this approach is that it is possible to compare more samples (labeled approaches are limited to the number of label the technique of choice supports) and one avoids problems that might arise from the labels influencing the fragmentation of the ions. The drawbacks is that sample preparation and other factors increase the amount of noise drowning out the signal.

Reference
Weisser, Hendrik; Nahnsen, Sven; Grossmann, Jonas; Nilse, Lars; Quandt, Andreas; Brauer, Hendrik; Sturm, Marc; Kenar, Erhan; Kohlbacher, Oliver; Aebersold, Ruedi; Malmstrom, Lars; e for high-throughput label-free quantitative proteomics. J Proteome Res (2013), -: Epub ahead of print.

Applications

MS is a powerful tool that can be used to learn much about biological systems. We regularily apply the technologies we have developed on real problems and this have lead to the publication of several scientific articles.

Reference
Malmstrom, Lars; Malmstrom, Johan; Aebersold, Ruedi; Quantitative proteomics of microbes: Principles and applications to virulence. Proteomics (2011), 11: 2947-56.
Nunn, Brook; Aker, Jocelyn; Shaffer, Scott; Tsai, Shannon; Strzepek, Robert; Boyd, Philip; Freeman, Theodore; Brittnacher, Mitchell; Malmstrom, Lars; Goodlett, David; Deciphering diatom biochemical pathways via whole-cell proteomics. Aquat Microb Ecol (2009), 55: 241-253.
Malmstrom, Lars; Hou, Liming; Atkins, William; Goodlett, David; On the use of hydrogen/deuterium exchange mass spectrometry data to improve de novo protein structure prediction. Rapid Commun Mass Spectrom (2009), 23: 459-461.
Goo, Young; Liu, Alvin; Ryu, Soyoung; Shaffer, Scott; Malmstrom, Lars; Page, Laura; Nguyen, Liem; Doneanu, Catalin; Goodlett, David; Identification of secreted glycoproteins of human prostate and bladder stromal cells by comparative quantitative proteomics. Prostate (2009), 69: 49-61.
Malmstrom, Erik; Sennstrom, Maria; Holmberg, Anna; Frielingsdorf, Helena; Eklund, Erik; Malmstrom, Lars; Tufvesson, Ellen; Gomez, Maria; Westergren-Thorsson, Gunilla; Ekman-Ordeberg, Gunvor; Malmstrom, Anders; The importance of fibroblasts in remodelling of the human uterine cervix during pregnancy and parturition. Mol Hum Reprod (2007), 13: 333-41.
Malmstrom, Johan; Larsen, Kristoffer; Malmstrom, Lars; Tufvesson, Ellen; Parker, Ken; Marchese, Jason; Williamson, Brian; Hattan, Steve; Patterson, Dale; Martin, Steve; Graber, Armin; Juhasz, H; Westergren-Thorsson, Gunilla; Marko-Varga, Gyorgy; Proteome annotations and identifications of the human pulmonary fibroblast. J Proteome Res (2004), 3: 525-37.
Malmstrom, Johan; Larsen, Kristoffer; Malmstrom, Lars; Tufvesson, Ellen; Parker, Ken; Marchese, Jason; Williamson, Brian; Patterson, Dale; Martin, Steve; Juhasz, Peter; Westergren-Thorsson, Gunilla; Marko-Varga, Gyorgy; Nanocapillary liquid chromatography interfaced to tandem matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry: Mapping the nuclear proteome of human fibroblasts. Electrophoresis (2003), 24: 3806-14.