With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel “multi-blind” spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data.

Redox-active cysteine, a highly reactive sulfhydryl, is one of the major targets of ROS. Formation of disulfide bonds and other oxidative derivatives of cysteine including sulfenic, sulfinic, and sulfonic acids, regulates the biological function of various proteins. We identified novel low-abundant cysteine modifications in cellular GAPDH purified on 2-dimensional gel electrophoresis (2D-PAGE) by employing selectively excluded mass screening analysis for nano ultraperformance liquid chromatography-electrospray-quadrupole-time of flight tandem mass spectrometry, in conjunction with MODⁱ and MODmap algorithm. We observed unexpected mass shifts (Δm = −16, −34, +64, +87, and +103 Da) at redox-active cysteine residue in cellular GAPDH purified on 2D-PAGE, in oxidized NDP kinase A, peroxiredoxin 6, and in various mitochondrial proteins. Mass differences of −16, −34, and +64 Da are presumed to reflect the conversion of cysteine to serine, dehydroalanine (DHA), and Cys-SO₂-SH respectively. To determine the plausible pathways to the formation of these products, we prepared model compounds and examined the hydrolysis and hydration of thiosulfonate (Cys-S-SO₂-Cys) either to DHA (Δm = −34 Da) or serine along with Cys-SO₂-SH (Δm = +64 Da). We also detected acrylamide adducts of sulfenic and sulfinic acids (+87 and +103 Da). These findings suggest that oxidations take place at redox-active cysteine residues in cellular proteins, with the formation of thiosulfonate, Cys-SO₂-SH, and DHA, and conversion of cysteine to serine, in addition to sulfenic, sulfinic and sulfonic acids of reactive cysteine.

Non-small cell lung cancer (NSCLC) is histologically classified into lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LSCC). However, some tumors are histologically ambiguous and other pathophysiological features or microenvironmental factors may be more prominent. Here we report integrative multiomics analyses using data for 229 patients from a Korean NSCLC cohort and 462 patients from previous multiomics studies. Histological examination reveals five molecular subtypes, one of which is a NSCLC subtype with PI3K-Akt pathway upregulation, showing a high proportion of metastasis and poor survival outcomes regardless of any specific NSCLC histology. Proliferative subtypes are present in LUAD and LSCC, which show strong associations with whole genome doubling (WGD) events. Comprehensive characterization of the immune microenvironment reveals various immune cell compositions and neoantigen loads across molecular subtypes, which predicting different prognoses. Immunological subtypes exhibit a hot tumor-enriched state and a higher efficacy of adjuvant therapy.

Identification of post-translational modifications (PTMs) is important to understanding the biological functions of proteins. MS/MS is a useful tool to identify PTMs. Most existing search tools are restricted to take only a few types of PTMs as input. Here we describe a new algorithm, called MODⁱ (pronounced “mod eye”), that rapidly searches for all known types of PTMs at once without limiting a multitude of modified sites in a peptide. MODⁱ introduces the notion of a tag chain, a combination structure made from multiple sequence tags, that effectively localizes modified regions within a spectrum and overcomes de novo sequencing errors common in tag-based approaches. MODⁱ showed its performance competence by identifying various types of PTMs in analysis of PTM-rich proteins such as glyceraldehyde-3-phosphate dehydrogenase and lens protein. We demonstrated that MODⁱ innovatively manages the computational complexity of identifying multiple PTMs in a peptide, which may exist in a greater variety than usually expected. In addition, it is suggested that MODⁱ has great potential to discover novel modifications.