Lars Malmstroem, Ph.D.

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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.

Research

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. One way of using a MS is to generate tertiary data that can be used to guide structure prediction and we reported one way of doing this here.

References