Mass spectrometry based proteomics

Mass spectrometry is routinely used to identify proteins, quantify their levels (relative or absolute) and to characterize protein post-translational modifications. Some useful reviews on the application of different mass spectrometry based approaches were written by Yates et al. [1, 2] and Cox et al. [3].

The advancements in the mass spectrometric technology, such as in instrument designs (dual pressure ion trap) and mass analyzers (orbitrap) have significantly improved the sensitivity and mass accuracy. Nevertheless, sample preparation and fractionation are very important especially when dealing with scarce clinical samples to be able to characterize high numbers of proteins and their post-translational modifications. Dealing with tissue samples or plasma poses additional challenges because of sample stability, solubility and abundance issues. Proteins in tissues are usually difficult to solubilize (which is critical for a successful tryptic digestion), whereas plasma contains abundant proteins (albumin) that can easily mask and thus prevent the identification of lower abundant proteins (such as signaling molecules and putative biomarkers). With the depletion of most abundant plasma proteins (up to 20), this approach then enables mass-spectrometry based proteomics to dig deeper into plasma proteome in order to identify low abundant proteins and their possible modifications.

The final goal of many proteomics experiments is to completely characterize a proteome: protein's identification, location, function, interacting partners, post-translational modifications (PTMs) and in many cases also the turnover rate of protein production/degradation or the rate of formation for a particular PTM. We would also like to know what are the occupancies for a particular PTM (e.g. what percentage of a given site in protein is phosphorylated). Some recent reviews on PTMs are the following: phosphorylation [4–6], acetylation [7], S-nitrosylation [8], glycosylation [9], ubiquitination [10], etc.

Relative protein quantitation as a tool in biomarker discovery

Mass spectrometry is an indispensible tool for relative protein quantitation which is usually applied to discovery of clinical biomarkers and mostly uses large-scale proteomics experiments. The first step in a typical large scale proteomics experiment is a separation technique on either the protein (e.g. 1D gel) or peptide level (e.g. SCX - strong cation exchange chromatography), followed by analysis of each of the individual fractions with liquid chromatography coupled to tandem mass spectrometry system (LC-MS/MS). The liquid chromatography is mostly reverse phase based with the flows in nanoliter range. Usually, 1D gel separation on protein level followed by an LC-MS/MS analysis gives a higher number of identified proteins because the first dimension separation is on the protein level as compared to SCX which separates peptides on the basis of charge. A summary of proteomics approaches using mass spectrometry is depicted in Figure 1.

The analysis of PTMs usually involves enrichment which is based on an antibody pull-down or it takes advantage of some physicochemical property of the PTM to selectively target proteins or peptides with that PTM. For example, tyrosine (Tyr) phosphorylated proteins (or peptides) are easily enriched by using a specific anti-pTyr antibody. On the other hand, serine or threonine phosphorylated proteins or peptides are enriched by using immobilized-metal affinity chromatography (IMAC) and metal-oxide affinity chromatography (e.g. TiO2 based). After the enrichment, the mass spectrometric analysis can pose some difficulties as well. A lot of PTMs are labile modifications that can easily fragment under collisionally induced dissociation in mass spectrometer [4].

There are several complementary approaches for relative protein quantification, such as DIGE (2D flourescence difference gel electrophoresis) [11], iTRAQ (isobaric tag for relative and absolute quantitation) [12] and SILAC (stable isotope labeling with amino acids in cell culture) [13], with DIGE being used the most in studies of fibrotic disorders.

Two-dimensional gels are an excellent way to separate proteins on the basis of their molecular weight and isoelectric point, enabling also visualization of some posttranslational modifications (such as phosphorylation). In the quantitative approach (DIGE), two protein samples are labeled with fluorescent dyes and when combined and separated on a 2D gel can be visualized by measuring the fluorescence showing whether a protein is up- or down regulated and for how much. The identification of individual spots of interest is carried out by mass spectrometry. The gels are usually scaled up (pick gel), stained with Coomassie blue, excised and digested with trypsin before being analyzed on a MALDI TOF/TOF or electrospray type of an instrument. Since fluorescence can be very sensitive, it is sometimes difficult to identify the proteins in spots by mass spectrometry which is a couple of orders less sensitive. Another drawback of this approach is that sometimes the individual spots may provide several identifications with similar proteins co-eluting.

As a complementary technique to DIGE, iTRAQ has recently gained a lot of popularity in the protein biomarker applications because of the ability to multiplex several samples in one run (up to 8) and because the number of identified proteins are usually higher.

Proteomic analysis of fibrotic samples

Proteomic analysis of fibrotic tissue is further complicated with the limited ability of most common techniques to solubilize proteins in a complicated matrix. Usually, the number of identified proteins from a tissue sample is smaller as compared to a cell lysate. Most of the biologically important proteins (such as proteins involved in signaling mechanisms) are of lower abundance, thus it is essential to identify as many proteins as possible. The other drawback associated with fibrotic tissue analysis is limitation with the quantitation techniques because some of them due to the nature of labeling cannot be applied (e.g. SILAC vs. iTRAQ). Plasma samples, on the other hand, pose challenges as well, mainly due to high abundant proteins that are usually depleted to enable identification of less abundant proteins in the sample.

Proteomics studies on organ fibrosis