Trimmed for Assembly
N-terminal proteomics and complexome profiling link ICP55 processing to the stability of mitochondrial protein assemblies.
A single-amino-acid difference at protein N termini can have proteome-wide consequences for mitochondrial complex stability, according to a recent study combining N-terminal proteomics and complexome profiling.
The study focused on intermediate cleaving peptidase 55 (ICP55), which acts after mitochondrial proteins are imported and their targeting sequences removed. ICP55 trims one additional N-terminal residue from a subset of proteins, but its substrate range and functional importance in human mitochondria have remained poorly defined.
Comparing wild-type and ICP55-deficient human cells using N-terminal proteomics, the researchers identified mature N termini for 446 processed mitochondrial proteins and 107 high-confidence ICP55 substrates. The analysis also uncovered previously unrecognized cleavage preferences involving arginine and glutamine, extending beyond motifs largely derived from yeast studies.
The consequences became clearer through stable isotope labeling by amino acids in cell culture (SILAC)-based complexome profiling. Mitochondrial protein assemblies were separated by blue-native PAGE into 44 fractions and analyzed by quantitative LC–MS/MS, producing migration profiles for 933 mitochondrial proteins. Loss of ICP55 significantly altered the profiles of 177, with large assemblies generally becoming less abundant and smaller complexes or monomeric forms appearing more frequently.
Respiratory chain complexes, the mitochondrial ribosome, metabolic enzymes, and the HSP60 chaperonin were among the affected assemblies. In HSP60, the team went further by producing proteins differing only in whether an additional N-terminal tyrosine was retained. The extra residue was sufficient to destabilize pre-assembled HSP60 complexes in vitro, supporting a direct link between incomplete processing and weakened protein-protein interactions.
By linking specific N-terminal proteoforms to the assembly states of hundreds of mitochondrial proteins, the study shows how a single-residue processing step can influence protein organization across an entire organelle.
Lipid Selectivity in the STARD Family
Native MS distinguishes endogenous phospholipid preferences across three STARD transporters and links phosphorylation to control of lipid transfer.
Native mass spectrometry has distinguished the endogenous lipid cargo of three closely related phospholipid transporters, revealing different preferences for the fatty-acid chains they carry and how phosphorylation can regulate lipid transfer.
STARD2, STARD7, and STARD10 shuttle phospholipids between cellular membranes, but their functional specialization has remained unclear. Most binding studies have relied on purified proteins and synthetic lipids, making it difficult to establish which species the transporters select from the much more competitive cellular lipidome.
The researchers, led by Carol Robinson, used multistage native MS to characterize the lipids carried by intact STARD protein–lipid complexes purified from human cells. All three proteins carried phosphatidylcholine (PC), but with distinct preferences for different acyl-chain compositions. STARD2 favored long, polyunsaturated PCs, while STARD10 preferentially carried di-unsaturated species. STARD7, by comparison, showed relatively little acyl-chain selectivity.
Those preferences were also reflected in changes to cellular lipid composition. Overexpressing STARD2 or STARD10 increased unsaturated PC species, whereas reducing STARD2 expression depleted polyunsaturated phosphatidylethanolamines. The effects extended to other phospholipids linked to PC synthesis, supporting a connection between transporter abundance and cellular acyl-chain profiles.
Native top-down MS identified phosphorylation of STARD2 and STARD10 in regions associated with membrane binding. Liposome assays then showed a functional consequence for STARD10: the phosphorylated protein was effectively inactive, while removing the phosphates restored lipid transfer.
Together, the measurements distinguish two ways in which closely related lipid transporters can shape lipid movement: selecting phospholipids according to acyl-chain composition and regulating when that cargo can be transferred. The authors now suggest testing whether these preferences translate into selective lipid transport in living cells, using probes with defined acyl chains to track the intracellular movement of preferred cargo.
Digging Into the Phosphoproteome
GoDig 2.0 repurposes discovery datasets into targeted libraries while increasing throughput for site-specific PTM quantification.
An updated targeted mass spectrometry platform, GoDig 2.0, has expanded high-throughput measurement of post-translational modifications, combining increased sample multiplexing with higher target success rates and streamlined assay development.
The update builds on GoDig’s automated targeting, which avoids manual scan scheduling and synthetic peptide standards, with faster data acquisition and compatibility with 35-plex tandem mass tags.
In a challenging benchmark containing both detectable and deliberately undetectable targets, GoDig 2.0 quantified around 911 peptide precursors per run, compared with 401 using its predecessor. With a more tractable 800-peptide target list, it quantified more than 99 percent in a single run across 35 multiplexed samples, corresponding to an effective throughput of around 370 samples per day.
The expanded targeting capacity was also applied to site-specific phosphorylation. Existing untargeted phosphoproteomics datasets were repurposed into a library covering 23,989 human phosphorylation sites, which avoided the need to build targeted libraries from scratch. Applied to three signaling pathways across five cell lines, GoDig 2.0 quantified site-level differences that varied substantially between cell types.
A pilot analysis of postmortem brain tissue illustrated the potential for more focused validation. Among samples from 10 people with Alzheimer’s disease and eight controls, untargeted analysis identified relatively few significant differences in tau phosphorylation. GoDig 2.0 identified significant differences in 23 of the 35 targeted tau phosphopeptides, including 11 that completely separated the Alzheimer’s and control groups in this small cohort. Among them was pTau217, an established Alzheimer’s biomarker.
The platform was also adapted to quantify ubiquitination and covalent compound engagement at reactive cysteines, indicating that its gains extend beyond phosphorylation. The authors now plan to expand the available target libraries and test the tau phosphorylation panel in larger cohorts and more accessible samples, including blood plasma.
A Plasmic Route to Hydrocarbon Analysis
Low-pressure plasma introduces controlled functional groups into hydrocarbons, converting poorly ionizing molecules into MS-friendly derivatives.
Hydrocarbons that are difficult to ionize directly can be made more amenable to mass spectrometry by chemically modifying them in a low-pressure plasma, with the reaction conditions controlling the functional groups introduced.
The approach, developed by Graham Cooks and colleagues at Purdue University, addresses the poor ionization of nonpolar hydrocarbons by converting them into more readily detected derivatives before MS analysis. A subatmospheric-pressure plasma provides stable discharges under mild conditions, allowing functionalization without extensive oxidation or fragmentation.
Under oxygenating conditions, the researchers generated ketones and unsaturated ketones, producing well-defined molecular ions with characteristic tandem MS fragmentation. The approach worked across linear, branched, cyclic, and aromatic hydrocarbons, while changing the reaction conditions could favor oxidation, dehydrogenation, or nitrogen insertion. In the latter case, hydrocarbons including cyclohexane and hexane formed nitrogen-containing derivatives whose MS/MS spectra showed characteristic ammonia loss.
To confirm that the oxygenated products reflected ketone formation, the team scaled up the plasma reaction to collect sufficient material for NMR analysis, which supported ketones as the dominant oxygen-insertion products. Phenylhydrazine derivatization of plasma-treated n-pentadecane provided additional evidence for carbonyl formation.
Rather than relying on direct ionization of chemically unresponsive hydrocarbons, the method makes controlled functionalization part of the analytical workflow, turning poorly ionizing molecules into derivatives that can be both detected and interrogated by tandem mass spectrometry.
(Mass) Spectacular and Strange
The Proteins Beneath the Paint
Ancient Egyptian artworks have long been studied for their colors, symbols, and surfaces. A new study turns to the less visible materials beneath: the proteins used to bind paint and glue objects together.
Researchers analyzed 28 microsamples from 14 painted ancient Egyptian artworks dating from 1425 BCE to 400 CE, including coffins, wall painting fragments, cartonnage, painted plaster, and limestone. After initial FTIR screening, the team used shotgun proteomics with nano-flow liquid chromatography tandem mass spectrometry to identify proteinaceous binders and adhesives.
Animal glue was common, with collagen peptides pointing mainly to cattle, but also to mixtures including sheep or goat, antelope relatives, donkey, and horse. The detection of COL3α1, associated with connective tissues, suggested that some glues were hide glues prepared from skin and related tissues rather than bone alone.
The more unexpected finding came from plants. Proteomics identified sesame and moringa seed-storage proteins in two artworks, while cereal proteins, mainly from wheat with barley and rye also detected, appeared in wall painting samples. Together with FTIR, GC-MS, and Py-GC-MS evidence, the results suggest that some plant proteins may reflect protein-rich by-products from seed or cereal processing rather than straightforward plant oils.
The study demonstrates how paleoproteomics can recover material choices that older approaches might miss, revealing binders and adhesives as clues to resource use, workshop habits, and symbolism in ancient Egyptian painting.
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