There are more than 200 Post-translational modifications (PTMs) known (see chapter 1 for more information). As a result of these diverse chemical modifications more than one million unique protein isoforms (proteoforms) arise from the human coding genome consisting of just around 21,000 genes (Figure 1). PTMs are valued as biomarkers useful in diagnosing and monitoring diseases and their treatments, while their underlying processes have been the targets for drug discovery. Realizing those opportunities depends on the availability of robust, reliable analytic tools, and mass spectrometry (MS ) is an emerging technology for this purpose.
Because PTMs affect the molecular weight of both intact proteins and protein fragments, MS allows the identification of specific PTMs on specific amino acid residues. MS interfaces nicely with centrifugation, electrophoresis, liquid chromatography, and many other front-end separation/purification techniques, so MS analysis is suited to many different sample types. A typical MS proteomic experiment will therefore seek PTM identification and location, as well as primary structural data.
Recent studies have uncovered more than 20,000 phosphorylation sites, 19,000 ubiquitination sites, and 3,600 acetylation sites on proteins commonly found in human cell lines. Given the molecular diversity of a typical proteomic sample, the presence of multiple PTM-isoforms for each protein, and the presence of background contaminants, most samples must undergo enrichment to improve MS detection. For example, phosphorylated peptides may be enriched with immobilized metal affinity chromatography, while acetylated or ubiquitinated peptides may be enriched through antibodies., Other peptide fractionation strategies include cation exchange, hydrophilic interaction liquid chromatography, stable-isotope labeling, and serial enrichment. All of these methods share the goal of increasing the concentration or accessibility of target post-translationally modified proteins in the presence of interfering species.
MS-based analysis of PTMs employs two major methods: top-down and bottom-up proteomics.
Conventionally, bottom-up proteomics digests proteins with proteolytic enzymes, separates them using chromatography or electrophoresis, and analyzes them by MS. Top-down methods use intact protein ions, often generated by electrospray ionization, which undergo gas-phase fragmentation. Some experts also refer to methods that purify intact proteins first, followed by digestion and MS analysis, as top-down. This definition emphasizes the purification step at the expense of mass spectrometry.
Depending on when the purification steps, if any, occur, methods utilizing proteolytic digestion may contain thousands or even tens of thousands of fragments from many different proteins, or the signature pieces of just a few proteins. For this reason many proteomics samples undergo two-dimensional purification, followed by peptide mass fingerprinting and tandem MS (MS–MS).
Bottom-up proteomics is relatively inefficient in terms of protein-sequence coverage, as only a small, variable fraction of the peptide fragments from any one protein is recoverable. This means that a significant quantity of PTM information is lost.
Since the top-down approach operates on intact proteins, structural characteristics that are lost in bottom-up MS are retained. Since the “starting material” is intact protein all fragmented species are relevant, and detection is possible for all PTMs. Eliminating the protein digestion step -- essentially allowing the MS instrument to do the heavy lifting -- also saves significant time.
Despite its advantages in some areas, the top-down approach is not a panacea. The technique will need to undergo additional improvement and refinement before it may be considered a robust approach to proteomics.
Intact proteins are, for one, more difficult to work with than peptides. Proteins tend to be less soluble than peptides, a factor affecting their suitability to purification by liquid chromatography. Large proteins, particularly membrane proteins, require detergents for solubilization. Common detergents, like sodium dodecyl sulfate (SDS), are incompatible with ESI, the ionization method of choice for very large, labile molecules.
Figure 1: Expansion of genes to protein proteoforms with PTMs
Sensitivity and detection limits of MS on proteins is lower, generally, than for peptides, and while less complex operationally the throughput of top-down is significantly lower than for bottom-up methods. Hence the top-down approach is used mostly for single, isolated proteins, protein mixtures of fairly low complexity, or proteins smaller than about 50 kDa.
Top-down proteomics is improving, however. Enhancements to enrichment/prefractionation methodology, MS instrumentation, and dissociation methods for intact-protein ions have made top-down MS analysis more accessible, particularly in translational medicine. For example, the top-down approach known as “spectrometric immunoassay” uses antibody-derivatized microcolumns to isolate intact proteins of interest as a front-end to MALDI-TOF MS analysis. Techniques such as these tend to automate top-down workflows, and will continue to support its adoption as an alternative to the bottom-up approach.
Investigators are also looking into middle-up and middle-down proteomics for unequivocal characterization of PTMs. Middle-up techniques break up the protein of interest by either stopping proteolytic digestion before it is complete, by chemically cleaving disulfide bonds, or through cyanogen bromide digestion of C-terminal methionine residues. Similarly, middle-down proteomics cleaves proteins into large fragments, but instead of direct MS analysis gas-phase dissociation is performed first.
MS has been indispensable for the growth in the PTM field, evident by the expansive number of proteoforms identified. However, functional characterization of the majority of these proteoforms is still unknown. Several different MS-based approaches aim to quantify PTM site stoichiometry, as a means to prioritize investigation of abundant proteoforms.18 Molecular biology tools and approaches may be useful to characterize promising proteoform targets. Likely, a combination of these approaches will be utilized to effectively characterize the next generation of proteoform biomarkers and therapeutic targets.
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