Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Global versus Targeted Analysis Strategies
Detection of PTMs by mass spectrometry can be achieved via global or targeted methods. The biological pathway of interest usually determines the type of PTM to be analyzed and associated methods. In a more targeted approach, researchers decide to investigate PTMs, because a protein of interest shows a higher than expected molecular weight or multiple bands by western blot after application of a stimulus, thus prompting speculation as to whether this could be due to PTM. Either way, the first step in PTM mapping is to determine the type of PTM of interest. In some cases the observed mass shift in a mass spectrometer indicates a certain PTM type. Many PTMs, however, result in the same mass addition (e.g., +42 Da for both acetylation and trimethylation). One powerful strategy in determining PTM identity involves the employment of the enzymes responsible for PTM removal. For example, after antibody enrichment of a modified protein, the antibody-bound protein can be incubated with general phosphatases, deubiquitinating enzymes (DUBs), or deSUMOylating enzymes (SENPs), and PTM removal can be assayed by western blot. Another method for PTM identification is western blotting with PTM specific “pan-antibodies.” Many commercially available antibodies exist for this purpose, recognizing common PTMs such as acetylation, methylation, ubiquitylation, and phosphorylation or even more rare PTMs such as crotonyl-, malonyl- or glutaryl-lysine modification. Once the type of PTM that is decorating a protein has been identified, the next step is to attempt to map the amino acid residue(s) that bear this modification.
One of the first applications of mass spectrometry in protein research was the mapping of a PTM on a single protein . A commonly used approach involves protein-level immunoprecipitation followed by separating the captured proteins by SDS-PAGE, excising the higher molecular weight band, and performing in-gel tryptic digestion followed by LC-MS/MS. By searching for mass shifts indicative of the suspected modification(s), PTM-containing peptides can be identified and the PTM site mapped back to the protein. The strategy of identifying proteins in complex mixtures by digesting them into peptides, sequencing the resulting peptides by tandem mass spectrometry (MS/MS), and determining peptide and protein identity through automated database searching is referred to as shotgun proteomics and is one of the most popular analysis strategies in proteomics . This protein-level enrichment approach, however, is dependent on sufficient levels of the modified protein compared to unmodified and the availability of protein-specific antibodies for immunoprecipitation. It is also possible that modifications may occur within the antibody epitope, blocking enrichment of the modified form altogether.
Researchers are commonly interested in analyzing PTMs from a complex mixture of proteins rather than on only one substrate. This can be a challenge, since modified peptides often occur in substoichiometric levels compared to unmodified versions and also may ionize less efficiently by electrospray ionization (ESI). However, several enrichment strategies exist, allowing for reduction of sample complexity and easier detection of the modified peptide species. Peptide-level immunoprecipitation using antibodies specific to a given PTM is an increasingly popular method of enrichment prior to MS. While this strategy can be employed for any PTM enrichment, it has been most commonly used for mapping ubiquitination sites. Tryptic digestion of ubiquitinated proteins generates a diglycine remnant attached to the ubiquitinated lysine residue (K-GG) that can be recognized by antibodies. The resulting mass shift of +114.0429 Da can be detected by MS/MS. Not only has K-GG peptide immu- noaffinity enrichment enabled the identification of hundreds of ubiquitination sites on a global level but it has also been shown to enhance identification of ubiquitination sites on individual proteins, when compared to protein-level IP coupled with MS/MS .
To understand the biological significance of a specific PTM, it is also important to determine the PTM site occupancy or percentage of a protein's total population that is modified. Quantification of site occupancy can be accomplished by combining antibody peptide enrichment with stable isotope-labeled internal standards of the same sequence, a method termed stable isotope standards and capture by anti-peptide antibodies (SISCAPA) . By coupling immunoprecipitation with stable isotope dilution multiple reaction monitoring (SID-MRM), absolute quantitation of both modified and unmodified protein populations can be determined in a high-throughput, multiplexing- compatible fashion .
In addition to antibody-based enrichment approaches, several strategies for chemical enrichment of PTM-containing subproteomes have been developed.
These approaches can also be coupled with the use of stable isotope standard peptides and SRM/MRM for accurate quantification of PTM dynamics. The most widely studied PTM, with the most variety of enrichment methods available, is phosphorylation. Global analysis of serine, threonine, and tyrosine phosphorylation can be achieved by a combination of peptide fractionation using strong cation exchange (SCX) followed by further enrichment with immobilized metal affinity chromatography (IMAC). The SCX/IMAC approach allows for enrichment of phosphorylated peptides to over 75% purity and ultimately identification of over 10,000 phosphorylation sites from 5 mg of starting protein [13, 19]. Another common approach for selective enrichment of the phosphoproteome is using metal oxide affinity chromatography (MOAC) such as titanium dioxide (TiO2)  or aluminum hydroxide (Al(OH3)) . MOAC methods have been reported to achieve higher sensitivity than IMAC (at the cost of lower specificity though). The combination of multiple enrichment approaches may ultimately be the best approach.
Phosphopeptide enrichment strategies can also be applied on crude protein extract to enrich for entire phosphoproteins. Enriched fractions are typically separated by two-dimensional gel electrophoresis (2D-GE) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In either case, each observed protein spot/band is quantified by its staining intensity, and selected spots/bands are excised, digested, and analyzed by MS. The advantage of phosphoprotein enrichment is that intact proteins are separated, and the molecular weight and isoelectric point of proteins can be determined. This greatly aids in protein identification by MS. However, protein-level enrichment has several disadvantages, including loss of small or hydrophobic proteins during precipitation steps, less specific enrichment when compared to phosphopeptides, and difficulty in identifying low-abundance proteins or modifications .
In summary, both targeted and global methods for PTM identification have been significantly tuned in recent years but are still facing challenges. The choice of method is usually dictated by the biological question. However, global strategies are becoming increasingly popular due to their versatility, sensitivity, and ability to collect a wealth of data, triggering new hypotheses that ask for validation by targeted experiments.
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