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 ~50% 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 high-throughput fashion.

Fig. 1.

Total ion chromatogram and several identifications from a GELFrEE fraction enriched for mitochondrial proteins. Using HCD, 153 proteins were identified in this single LC-MS/MS run.

Fig. 2.

Analysis of the 1,220 total proteins identified in this study at a 1% FDR. A, distribution of the identifications between GELFrEE of enriched mitochondrial proteins, IEF fractions, and other GELFrEE preparations at 5% and 1% FDRs. B, subcellular localization of the identified proteins revealed 347 mitochondrial proteins identified along with a significant number of proteins in most of the common organelles. C, distribution of all annotated human mitochondrial proteins as a function of protein length overlaid with those identified in this work.

Fig. 3.

Distribution of the number of transmembrane helices predicted for the 301 integral membrane proteins identified in this study. Proteins with between 1 and 12 transmembrane domains were identified, with 48% containing 2 or more.

Fig. 4.

Detection of multiply modified proteins. A, phosphorylation of protein transport protein sec61 β (P60468), which was also observed with a +58 Da form, also phopsphorylated. B, NADH dehydrogenase 1 β subcomplex subunit 3 was detected with two histidine methylations along with the unmodified form, with the singly methylated form being the most abundant.

Fig. 5.

Identification of post-translational modifications and sequence variants. A, signal peptidase complex subunit 1 (Q9Y6A9) was found as a shortened form, with the predicted Met68 as the initial methionine. B, Ras-related protein Rap-1b (P61224) showed cleavage of its three C-terminal residues and methylation of the C terminus. C, Ras-related protein Rab-11a (P62491) exhibited geranylgeranylation on two adjacent cysteine residues as well as cleavage of the initial methionine and acetylation of the N terminus.

Fig. 6.

Comparison of untreated and senescent H1299 cells at the protein identification and proteoform levels. A, Venn diagram displaying the protein identifications found in the two cell states. B, detection of HMGA2 in H1299 control and senescent cells. HMGA2 was detected through GELFrEE-LC-MS and showed significantly lower abundance than HMGA1. C, the phosphorylated proteoforms of HMGA2 are dynamic during senescence, with forms containing up to five phosphorylations visible (lower panel). The smaller peaks to the right of each major peak represent oxidation (+16), not methylation (+14).