Analysis of intact proteins, termed top-down proteomics, has the potential to provide comprehensive profiling of proteoforms particular since the connectivity of multiple PTM is retained. While bottom-up analysis for identification and relative quantification provides a catalog of post-translationally modified sites, information on PTM combinations at the proteoform level is lost, particularly for sites in different regions of the protein. Intact proteins are amenable to MS, particularly since the occurrence of PTMs has a much smaller effect on the ionization/detection efficiency of intact proteins relative to peptides .
A typical top-down workflow employs accurate mass determination of the intact protein at the MS level, followed by primary fragmentation and fragment mass MS2 analysis. Isolation of individual fragments for further MS3 fragmentation builds up amino acid sequence and PTM information . The technique is typically applied to a single protein and requires mass resolution and software for deconvolution of the isotopic peaks to determine the charge states and calculate precursor and product ion masses from complex spectra.
A major technical breakthrough has been the ability to resolve intact proteins to reduce sample complexity for analysis by a top-down approach . Proteins were fractionated by in solution isoelectric focusing (slEF) followed by gel-eluted liquid fraction electrophoresis GELFrEE. Isoelectric focusing separates post-translationally modified proteoforms since acetylation and methylation act to neutralize charge relative to the unmodified protein. Combining slEF with molecular weight separation resolves proteoforms, in a manner analogous to 2D gel separation, but instead in liquid phase enabling recovery of intact proteins for resolution by LC-MS analysis. Protein identification was achieved from m/z values of intact mass and matching MS2 fragments from the N- and C-termini . The presence of a PTM on intact proteins can be deduced by observing an increase in the precursor mass and a shift in the masses of the fragment ions containing the PTM. This study generated proof of concept of being able to identify pairs of protein intact mass values with mass differences consistent with mono-, di-, and trimethylation, acetylation in addition to mono- and diphosphorylated proteins in a complex mixture. A total of 3093 proteoforms corresponding to 1043 proteins were identified, for which 538 lysine acetylations and 158 methylations were assigned. The use of SDS in the GELFrEE system-enabled analysis of membrane proteins and proteins up to 80 kDa were evaluated. The method is time consuming, requiring two rounds of offline fractionation and multiple frac- tions/sample from which SDS must be removed for compatibility with LC-MS analysis, but achieves significant coverage of the proteome for proteome wide monitoring using 0.5-1 mg starting material of cell lysates or mitochondrial fractions .
A technically simpler, but high-resolution protein separation employing two-dimensions of HPLC separation, couples hydrophobic interaction chromatography (HIC) to orthogonal RP-HPLC to provide an alternative separation method for top-down proteomic analysis . The use of MS compatible ammonium tartrate buffer for the HIC separation results in separation orthogonal to that of RP-HPLC. The method was evaluated by analysis of mixes of 4 or 10 protein standards and an Escherichia coli lysate providing proof of principle of applicability to top-down proteomics. The study was not PTM directed but has potential applicability to the analysis of acetylated and methylated proteoforms.
The middle-down approach analyzes longer peptides (3000-10,000 Da), which are typically generated using nontryptic methods, for example, alternative enzymes such as Glu-C, Asp-N, OmpT, or chemical cleavage. The technique combines the benefits of being a relatively simple workflow compared with top-down analysis with a greater ability to map combinatorial modifications than bottom-up peptide-centric approaches. In terms of fragmentation methods, middle-down approaches have been used with both CID  and ETD . ETD is widely employed due to applicability to higher charge states and generation of fragments with sequencing and acetylation and methylation site assignment potential . Step change in method utility comes from implementation of combined ETD-PRM for targeted analysis of Histone H3 acetylation and methylation variants .