Molecular & Cellular Proteomics最新文献

Translational errors (TEs) result in a mismatch between mRNA codons and the amino acids (AAs) of the corresponding protein. Unlike DNA mutations or RNA editing, where nucleotide sequences can be used to infer AA substitutions, TEs can only be detected at the protein level. Although high-throughput mass spectrometry (MS) proteomics offers the potential to resolve peptide sequences and could theoretically be used to identify TEs, the feasibility of current MS data analysis approaches for this application remains uncertain. Here, we utilize patient-derived xenograft proteomics data, which include both human and mouse peptides with identifiable cross-species AA variations, as a ground truth for benchmarking TE identification methods. By using high-confidence mouse peptides as surrogates for "TE-containing" peptides, we show that current open search approaches can achieve >65% overall sensitivity and >70% overall precision for high-quality samples. The intersection of different search strategies significantly enhances precision, albeit at the expense of reduced sensitivity. Notably, the evaluation metrics vary significantly across individual AA substitutions, suggesting that caution is warranted when detecting or interpreting specific AA substitutions. Moreover, closed searches targeting predefined AA changes exhibit poor precision, with post-translational modification mislocalization identified as a key bottleneck for this application. Overall, our study provides a first-of-its-kind benchmark for MS-based TE discovery and offers guidance for optimizing MS search strategies.

Malaria transmission from humans to mosquitoes is essential to the parasite life cycle. In the human malaria parasite, Plasmodium falciparum, the rate of commitment to produce the sexual transmission stages, or gametocytes varies and is governed by genetic, epigenetic and environmental factors. The sexually committed parasite has so far remained elusive due to the lack of markers to efficiently isolate these parasites for subsequent functional studies including proteomic analysis of the isolated population. Here, we demonstrate that MSRP1 is a highly specific sexual commitment marker. Using this marker, we generated and validated reporter parasite lines for subsequent FACS-based isolation of sexually and asexually committed parasites. Proteomics of isolated parasites defined distinct protein signatures, including several merozoite surface proteins, indicating functional differences between the two parasite populations. This study provides a blueprint for systematic characterization of the parasite stage at this crucial juncture in the life cycle.

High-throughput proteomics is critical for understanding biological processes, enabling large-scale studies such as biomarker discovery and systems biology. However, current mass spectrometry technologies face limitations in speed, sensitivity, and scalability for analyzing large sample cohorts. The Thermo Scientific Orbitrap Astral Zoom mass spectrometer (MS) was developed to address these limitations by improving acquisition speed, ion utilization, and spectral processing, which are all essential for advancing proteome depth in high-throughput proteomics. The Orbitrap Astral Zoom MS achieves ultra-fast MS/MS scan rates of up to 270 Hz with enhanced ion utilization through pre-accumulation, enabling the identification of ∼100,000 unique peptides and >8400 proteins in a single 300 samples-per-day analysis of human cell lysate. The optimized system reduces analysis time by 40%, achieves near-complete proteome coverage (>12,000 proteins) in 2.7 h, and enables ultra-high-throughput workflows, identifying >7000 proteins in a 500 samples-per-day method with exceptional reproducibility (pairwise Pearson correlations >0.99). These advancements establish the Orbitrap Astral Zoom MS among the fastest and most sensitive instruments under the tested conditions, significantly enhancing speed, sensitivity, and scalability, paving the way for routine large-scale proteomics with applications in clinical research and systems biology.

Alzheimer's disease (AD) is an age-associated neurodegenerative disorder characterized by amyloid plaques, tau hyperphosphorylation, and synaptic dysfunction. Most available cellular AD models lack aging features, limiting their ability to recapitulate key pathological mechanisms. Here we applied high-resolution mass spectrometry-based multiplexed proteomics and phosphoproteomics in a discovery setting to characterize an accelerated AD (acAD) model that combines amyloid precursor protein (APP) and presenilin (PSEN) mutations with progerin, an aging-associated Lamin A mutant that accelerates aging. Across four phenotypes (control, progerin, classic AD, and acAD), we identified 8279 proteins, quantified 6081 proteins, and detected phosphorylation dynamics. Relative to the classic model, acAD exhibited broader proteome remodeling, including amplified downregulation of synaptic and cytoskeletal proteins, upregulation of transcription and translation machinery, and pathway-level changes in neuronal signaling, mitochondrial dynamics, and neuroinflammation. Phosphoproteome analysis revealed widespread changes in RNA-binding and cytoskeletal proteins, aligning with recent data from two murine AD models. These findings show that acAD captures canonical AD phenotypes while uniquely modeling age-related inflammation and phosphorylation, providing a resource to accelerate studies of proteome-level mechanisms of AD progression and to inform strategies targeting cytoskeletal and inflammatory pathways.

Membrane proteins (MPs) are vital to cellular signaling, metabolism, and disease pathology, yet remain underrepresented in proteomics. To address this, several independent workflows have been developed to enable the profiling of the membrane proteome; however, the relative advantages and limitations of each method remain poorly defined. Here, we systematically compare four classical solid-phase membrane proteomic workflows (SP3, SP4, FASP, S-Trap) and three membrane mimetic strategies (Peptidisc, nanodisc, and SMALP copolymer) for mass spectrometry-based membrane proteome profiling, using healthy (LFD) and obese (HFD) mouse liver tissue. We found that the solid-phase methods yield higher total protein identifications, while the membrane mimetic systems enrich MPs. SMALP copolymer displays intermediate characteristics between the solid-phase and membrane mimetic workflows. Peptidisc and nanodisc stand out for their enrichment of MPs, although Peptidisc shows better enrichment of plasma membrane integral MPs, particularly those with 11+ transmembrane segments. In the context of HFD-induced liver proteome remodeling, the Peptidisc workflow outperformed the other six methods by capturing the highest number of differentially expressed MPs and demonstrating the lowest standard deviation of MP-level dysregulation. Collectively, this comparative analysis highlights the trade-offs between depth of proteome coverage and MP enrichment across workflows, underscoring the importance of method selection based on total protein counts, MP enrichment, and the precise detection of MP-level dysregulation.

We present the first integrated transcriptomic and proteomic profiling of the iridocorneal region in the spontaneous murine glaucoma model DBA/2J and DBA/2J-Gpnmb⁺/Sj controls to define molecular changes associated with ocular hypertension and glaucoma. Using RNA sequencing and label-free quantitative proteomics, we identified over 20,000 transcripts and 8500 proteins, creating a comprehensive molecular atlas of glaucoma-related alterations in DBA/2J mice. Principal component and differential expression analyses revealed distinct genotype-specific molecular signatures. In DBA/2J mice, upregulated genes were enriched in pathways related to extracellular matrix remodeling, collagen organization, TGF-β signaling, and inflammation. Proteomic data confirmed increased levels of complement components, antigen presentation proteins, and autophagy markers. Integrated analyses identified 29 genes upregulated at both transcript and protein levels, primarily involved in extracellular matrix structure and immune regulation. Downregulated genes were associated with melanocyte differentiation and pigment-organelle function, including Pmel, a gene implicated in pigmentary glaucoma. Cross-referencing with human genome-wide association studies data revealed overlap with glaucoma-associated genes (LTBP2, LOXL1, COL11A1, VCAM1), alongside reduced expression of Angpt and Lmx1b, linked to ocular hypertension. Together, these findings support the existence of an immune-fibrotic feed-forward loop and implicate collagen-elastic fiber dysfunction as a central mechanism in glaucoma pathogenesis.

S-palmitoylation is a poorly understood post-translational modification that is gaining more attention as an essential regulator of cellular processes. The reversible nature of S-palmitoylation may allow for fine-tuned control of cellular events and adaptation to stimuli. The detection of S-palmitoylated proteins and peptides includes the Acyl-Biotin Exchange (ABE) method, Acyl resin-assisted Capture (Acyl-RAC), metabolic labelling, and derivatives thereof. We present a novel method of enrichment of S-palmitoylated peptides termed SDC Acid Precipitation Enrichment (SDC-ACE). Here, S-palmitoylated peptides are enriched by taking advantage of their co-precipitation with Sodium deoxycholate (SDC) under acidic conditions, allowing easy and fast separation of lipidated peptides from the sample suspension. We initially applied our novel method for the characterization of the mouse brain, providing an in-depth analysis of S-palmitoylation events within the brain and comprehensive profile of the mouse brain S-palmitoylome. Further, we applied our method for mapping mouse tissue-specific S-palmitoylation, highlighting the extensive role of S-palmitoylation throughout various organs in the body. Finally, we applied our methods for studying the brain palmitoylome of diabetic db/db mouse, uncovering alterations in the palmitoylation of proteins associated with obesity and type 2 diabetes. The SDC-ACE method allows fast and easy enrichment of S-palmitoylated peptides, providing a valuable tool for exploring the dynamics and function of S-palmitoylation in diverse biological systems.

In recent years, arboviral infections have surged dramatically because of the geographic expansion of Aedes and Culex mosquitoes, their main vector mosquitoes. Despite significant efforts to uncover arbovirus-host interactions and viral protein effector functions in mammals, systematic studies aiming to characterize virus-vector interactions in arthropods are largely missing, and the functions and cellular targets of many arboviral proteins in mosquitoes remain elusive. Here, we applied a multiomic approach to systematically evaluate the ability of arboviral capsids to interact with the Aedes aegypti proteome. This extensive multimodal atlas across 11 pathogenic arboviral species spanning three viral genera revealed shared and distinct host factor specificities, uncovering species-, genus-, and vector preference-specific patterns of host usage in mosquitoes. Functional phenotypic screening of 110 newly discovered host proteins across three prototypic arboviruses (La Crosse virus, dengue virus, and West Nile virus) identified several novel host dependency factors, including a new role for the chromatin-remodeling Brahma complex in orthoflavivirus replication. Using a combination of biochemical and sequencing approaches, we characterized the cellular determinants of these interactions and profiled their functional consequences on the chromatin landscape. Altogether, this study provides a multilayered repository to categorize and characterize arboviral capsid effector functions in invertebrates, providing important cues on novel mechanisms of transcriptional regulation via capsid-mediated modulation of chromatin accessibility in insects.

Integrins are key plasma membrane proteins that mediate cell-ECM adhesion and communication, and they rely on a conformational change for their activation and bidirectional signaling. However, there are few in vivo studies of integrin activation. Here, we identify Integrin α5 (Itgα5)-associated proteins in the physiological setting of zebrafish somite morphogenesis. Using label-free mass spectrometry, we compared Itgα5-associated proteins in different integrin activation states. As expected, we found active Itgα5 enriched extracellular matrix (ECM) proteins. Surprisingly, inactive Itgα5 incapable of binding ligand recruits actin cytoskeletal proteins as efficiently as the active integrin. We validated Itgα5's linking to actin adaptors using Parallel Reaction Monitoring (PRM). We then focused on α-actinin 4 (Actn4), an actin cross-linker, which we find preferentially associates with inactive Itgα5. Along zebrafish somite boundaries, Itgα5 and Actn4 displayed on and off co-localization, and Actn4 showed a stronger correlation with wild-type and inactive Itgα5 compared with the active Itgα5. We also found that deleting the actin-binding domain (Actn4^ABDdel) resulted in cytoplasmic retention and loss of colocalization with Itgα5. These findings suggest that Itgα5 and Actn4 cooperate during somite boundary formation and that actin cytoskeleton reorganization facilitates their colocalization. Furthermore, we showed ligand-binding-deficient Itgα5 associated with Paxillin a (Pxna), a scaffold protein highly enriched at somite boundaries and strongly correlated with activated Itgα5. This study provides novel insights into in vivo integrin activation and integrin-actin interactions and broadens our understanding of integrin's role in tissue morphogenesis. Data are available via ProteomeXchange with identifiers PXD024942, PXD065495, PXD058516, PXD058550, and PXD058747.

The MIRAGE (Minimum Information Required for A Glycomics Experiment) guidelines for mass spectrometry (MS) data were initially developed to standardize the reporting of instrumentation, data acquisition and analytical details of the MS-based identification of released glycans. However, the growing interest in the study of intact glycoproteins and recent advances in MS-based glycoproteomics now necessitate a revision and expansion of these guidelines. This update includes an enhanced section focused on glycan structure analysis (glycomics) and introduces a new component tailored to the specific requirements of glycoproteomics. It addresses both shared and unique aspects of each approach and highlights glycoinformatics resources designed to facilitate data submission in compliance with the updated standards.