Large-scale Top-down Proteomics of the Human Proteome: Membrane Proteins, Mitochondria, and Senescence

Abstract

Top-down proteomics is emerging as a viable method for the routine identification of hundreds to thousands of proteins. In this work we report the largest top-down study to date, with the identification of 1,220 proteins from the transformed human cell line H1299 at a false discovery rate of 1%. Multiple separation strategies were utilized, including the focused isolation of mitochondria, resulting in significantly improved proteome coverage relative to previous work. In all, 347 mitochondrial proteins were identified, including ϳ50% of the mitochondrial proteome below 30 kDa and over 75% of the subunits constituting the large complexes of oxidative phosphorylation. Three hundred of the identified proteins were found to be integral membrane proteins containing between 1 and 12 transmembrane helices, requiring no specific enrichment or modified LC-MS parameters. Over 5,000 proteoforms were observed, many harboring post-translational modifications, including over a dozen proteins containing lipid anchors (some previously unknown) and many others with phosphorylation and methylation modifications. Comparison between untreated and senescent H1299 cells revealed several changes to the proteome, including the hyperphosphorylation of HMGA2. This work illustrates the burgeoning ability of top-down proteomics to characterize large numbers of intact proteoforms in a highthroughput fashion. Molecular & Cellular Proteomics

Top-down proteomics: applications, recent developments and perspectives

“…Top-down proteomic analysis of H1299 human cancer cell line proteome using this instrument has also been reported and identified 690 unique proteins from the H1299 human cancer cell line [88]. Similarly, top-down analysis and the identification of 1976 unique proteins from an H1299 cell line has also been reported [89]. Other types of mass spectrometry instruments (e.g., time-of-flight mass spectrometers and ion-traps) can also be applied for intact proteins analysis, but till date they are less effective than FTICRs, and hybrid linear ion trap (LTQ)-orbitraps [78] or LTQ-FTICRs [90].…”

“…Other types of mass spectrometry instruments (e.g., time-of-flight mass spectrometers and ion-traps) can also be applied for intact proteins analysis, but till date they are less effective than FTICRs, and hybrid linear ion trap (LTQ)-orbitraps [78] or LTQ-FTICRs [90]. In fact, the new-design orbitrap from Thermo Fisher utilizes a higher field orbitrap analyzer and improved ion-trap, providing another opportunity for top-down proteomics [89]. The combination gives improved resolving power over older designs and higher speed, making it competitive with FTICR-based instruments [91].…”

Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics

“…Searches were performed using an iterative, three-tiered, absolute mass search tree (2.5 Da, 200 Da, and 2000 Da intact mass tolerance), and then re-searched using a biomarker search with a 15 ppm tolerance for intact protein mass and a 10 ppm mass tolerance for fragment ions. In order to estimate the false discovery rate (FDR) for each data set, spectra were searched against a scrambled database and their p-scores derived from a Poisson statistical model (37) were fit to a gamma distribution as described previously (16,38). The E-values corresponding to a 5% FDR cutoff for absolute mass searches are as follows: GELFrEE, 1.1 ϫ 10 Ϫ6 ; whole venom, 1.7 ϫ 10 Ϫ6 ; sIEF 2.7 ϫ 10 Ϫ6 .…”

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…For this approach to become more widely used in solving biological problems, methods are needed to improve the characterization of RNA mixtures -where a goal of characterizing the complete cellular mixture of RNAs could be foreseen in a manner similar to that reported in proteomics. 120 Here, as with the bottom-up strategy, improvements in analyte separation prior to mass spectrometry will be key. In closing, it is important to keep in mind the particular advantages and disadvantages of any analytical technique.…”