Mass Spectrometric Analysis Methods for the Detection of PTMs
Mass spectrometers are powerful, analytical tools that have evolved rapidly over the past few decades to become the instrument of choice for protein and peptide characterization. Mass spectrometry is often used in parallel to other techniques such as western blot analysis or protein microarrays for detecting and quantifying PTMs. One of the main advantages of mass spectrometry is the ability to rapidly analyze many samples in a high-throughput manner. Mass spectrometric analyses can be divided into three main strategies: “bottom-up" “middle-down" and “top-down” proteomic approaches . Laboratories typically employ bottom-up proteomic methodologies to characterize PTMs. Proteins of interest are purified and proteolytically digested with an enzyme such as trypsin, with resultant peptides being separated by reversed-phase chromatography or another analytical method compatible with mass spectro- metric analysis. One of several fragmentation methods and ion detection methodologies can then be employed (see Sections 1.3.1-1.3.4 for description of the various types of bottom-up proteomic analyses). It is common to associate “data-dependent” MS/MS analysis with bottom-up approaches, where resulting peptide spectra are then pieced back together in silico to give an overview of the protein and its PTMs.
In top-down proteomics, intact protein ions or large protein fragments are subjected to gas-phase fragmentation for MS analysis. Here, a variety of fragmentation mechanisms can be employed to induce dissociation and mass spectrometric analysis of the protein including collision-induced dissociation (CID), electron transfer dissociation (ETD), and electron capture dissociation (ECD) [24-26]. High-resolution mass detectors such as the quadrupole-time of flight (Q-TOF), Fourier transform ion cyclotron resonance (FT-ICR), or orbitrap mass spectrometers are typically employed as the spectra generated from top-down fragmentation tend to be highly charged and therefore difficult to resolve without high-resolution power. Top-down proteomics to date has been a less popular tool for characterizing PTMs than bottom-up analysis. However, it is an invaluable tool in cases where a bottom-up approach would lose contextual information about combinatorial PTM distribution (e.g., in the case of histone PTM analysis ). The middle-down approach has more commonly been employed as a strategy whereby a proteolytic enzyme can be used to generate longer polypeptides from a protein of interest and has shown utility in analyzing complex PTMs such as the histone code [28, 29]. Compared to middle-down and top-down methods, the bottom-up approach often offers better front-end separation of peptides, typically equating to higher sensitivity and selectivity. There are however some limitations to the bottom-up approach including the risk of low sequence coverage, particularly when employing a single proteolytic enzyme such as trypsin where cleavage may result in peptides yielding chemophysical properties with poor analytical attributes, such as size or substandard hydrophobicity.