SECRET AGENT O-GlcNAcylates Hundreds of Proteins Involved in Diverse Cellular Processes in Arabidopsis.

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.

Differential Analysis of Cereblon Neosubstrates in Rabbit Embryos Using Targeted Proteomics.

Targeted protein degradation is the selective removal of a protein of interest through hijacking intracellular protein cleanup machinery. This rapidly growing field currently relies heavily on the use of the E3 ligase cereblon (CRBN) to target proteins for degradation, including the immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide which work through a molecular glue mechanism of action with CRBN. While CRBN recruitment can result in degradation of a specific protein of interest (e.g., efficacy), degradation of other proteins (called CRBN neosubstrates) also occurs. Degradation of one or more of these CRBN neosubstrates is believed to play an important role in thalidomide-related developmental toxicity observed in rabbits and primates. We identified a set of 25 proteins of interest associated with CRBN-related protein homeostasis and/or embryo/fetal development. We developed a targeted assay for these proteins combining peptide immunoaffinity enrichment and high-resolution mass spectrometry and successfully applied this assay to rabbit embryo samples from pregnant rabbits dosed with three IMiDs. We confirmed previously reported in vivo decreases in neosubstrates like SALL4, as well as provided evidence of neosubstrate changes for proteins only examined in vitro previously. While there were many proteins that were similarly decreased by all three IMiDs, no compound had the exact same neosubstrate degradation profile as another. We compared our data to previous literature reports of IMiD-induced degradation and known developmental biology associations. Based on our observations, we recommend monitoring at least a major subset of these neosubstrates in a developmental test system to improve CRBN-binding compound-specific risk assessment. A strength of our assay is that it is configurable, and the target list can be readily adapted to focus on only a subset of proteins of interest or expanded to incorporate new findings as additional information about CRBN biology is discovered.

optiPRM: A Targeted Immunopeptidomics LC-MS Workflow With Ultra-High Sensitivity for the Detection of Mutation-Derived Tumor Neoepitopes From Limited Input Material.

Personalized cancer immunotherapies such as therapeutic vaccines and adoptive transfer of T cell receptor-transgenic T cells rely on the presentation of tumor-specific peptides by human leukocyte antigen class I molecules to cytotoxic T cells. Such neoepitopes can for example arise from somatic mutations and their identification is crucial for the rational design of new therapeutic interventions. Liquid chromatography mass spectrometry (LC-MS)-based immunopeptidomics is the only method to directly prove actual peptide presentation and we have developed a parameter optimization workflow to tune targeted assays for maximum detection sensitivity on a per peptide basis, termed optiPRM. Optimization of collision energy using optiPRM allows for the improved detection of low abundant peptides that are very hard to detect using standard parameters. Applying this to immunopeptidomics, we detected a neoepitope in a patient-derived xenograft from as little as 2.5 × 106 cells input. Application of the workflow on small patient tumor samples allowed for the detection of five mutation-derived neoepitopes in three patients. One neoepitope was confirmed to be recognized by patient T cells. In conclusion, optiPRM, a targeted MS workflow reaching ultra-high sensitivity by per peptide parameter optimization, makes the identification of actionable neoepitopes possible from sample sizes usually available in the clinic.

prM-reactive antibodies reveal a role for partially mature virions in dengue virus pathogenesis.

Cleavage of the flavivirus premembrane (prM) structural protein during maturation can be inefficient. The contribution of partially mature flavivirus virions that retain uncleaved prM to pathogenesis during primary infection is unknown. To investigate this question, we characterized the functional properties of newly-generated dengue virus (DENV) prM-reactive monoclonal antibodies (mAbs) in vitro and using a mouse model of DENV disease. Anti-prM mAbs neutralized DENV infection in a virion maturation state-dependent manner. Alanine scanning mutagenesis and cryoelectron microscopy of anti-prM mAbs in complex with immature DENV defined two modes of attachment to a single antigenic site. In vivo, passive transfer of intact anti-prM mAbs resulted in an antibody-dependent enhancement of disease. However, protection against DENV-induced lethality was observed when the transferred mAbs were genetically modified to inhibit their ability to interact with Fcγ receptors. These data establish that in addition to mature forms of the virus, partially mature infectious prM+ virions can also contribute to pathogenesis during primary DENV infections.

Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice.

One of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/- mice were subfertile, whereas Prm1-/- mice were infertile. Prm1-/- and Prm2-/- sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/- sperm displayed only moderate DNA damage. The majority of Prm1+/- sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/- sperm. However, sperm from Prm1+/- and Prm1-/- mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/- animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

Targeted Proteomic Quantitation of NRF2 Signaling and Predictive Biomarkers in HNSCC.

The NFE2L2 (NRF2) oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion, and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry-based targeted proteomics assay based on internal standard-triggered parallel reaction monitoring to quantify 69 NRF2 pathway components and targets, as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved an existing internal standard-triggered parallel reaction monitoring acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded HNSCCs, NRF2 signaling intensity positively correlated with NRF2-activating mutations and with SOX2 protein expression. Protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus infection status. CDKN2A (p16) protein expression positively correlated with the human papilloma virus oncogenic E7 protein and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or formalin-fixed paraffin-embedded archived tissues in under 90 min.

The First Pituitary Proteome Landscape From Matched Anterior and Posterior Lobes for a Better Understanding of the Pituitary Gland.

To date, very few mass spectrometry (MS)-based proteomics studies are available on the anterior and posterior lobes of the pituitary. In the past, MS-based investigations have focused exclusively on the whole pituitary gland or anterior pituitary lobe. In this study, for the first time, we performed a deep MS-based analysis of five anterior and five posterior matched lobes to build the first lobe-specific pituitary proteome map, which documented 4090 proteins with isoforms, mostly mapped into chromosomes 1, 2, and 11. About 1446 differentially expressed significant proteins were identified, which were studied for lobe specificity, biological pathway enrichment, protein-protein interaction, regions specific to comparison of human brain and other neuroendocrine glands from Human Protein Atlas to identify pituitary-enriched proteins. Hormones specific to each lobe were also identified and validated with parallel reaction monitoring-based target verification. The study identified and validated hormones, growth hormone and thyroid-stimulating hormone subunit beta, exclusively to the anterior lobe whereas oxytocin-neurophysin 1 and arginine vasopressin to the posterior lobe. The study also identified proteins POU1F1 (pituitary-specific positive transcription factor 1), POMC (pro-opiomelanocortin), PCOLCE2 (procollagen C-endopeptidase enhancer 2), and NPTX2 (neuronal pentraxin-2) as pituitary-enriched proteins and was validated for their lobe specificity using parallel reaction monitoring. In addition, three uPE1 proteins, namely THEM6 (mesenchymal stem cell protein DSCD75), FSD1L (coiled-coil domain-containing protein 10), and METTL26 (methyltransferase-like 26), were identified using the NeXtProt database, and depicted tumor markers S100 proteins having high expression in the posterior lobe. In summary, the study documents the first matched anterior and posterior pituitary proteome map acting as a reference control for a better understanding of functional and nonfunctional pituitary adenomas and extrapolating the aim of the Human Proteome Project towards the investigation of the proteome of life.

A semi-automated workflow for DIA-based global discovery to pathway-driven PRM analysis.

Targeted proteomics, which includes parallel reaction monitoring (PRM), is typically utilized for more precise detection and quantitation of key proteins and/or pathways derived from complex discovery proteomics datasets. Initial discovery-based analysis using data independent acquisition (DIA) can obtain deep proteome coverage with low data missingness while targeted PRM assays can provide additional benefits in further eliminating missing data and optimizing measurement precision. However, PRM method development from bioinformatic predictions can be tedious and time-consuming because of the DIA output complexity. We address this limitation with a Python script that rapidly generates a PRM method for the TIMS-TOF platform using DIA data and a user-defined target list. To evaluate the script, DIA data obtained from HeLa cell lysate (200 ng, 45-min gradient method) as well as canonical pathway information from Ingenuity Pathway Analysis was utilized to generate a pathway-driven PRM method. Subsequent PRM analysis of targets within the example pathway, regulation of apoptosis, resulted in improved chromatographic data and enhanced quantitation precision (100% peptides below 10% CV with a median CV of 2.9%, n = 3 technical replicates). The script is freely available at https://github.com/StevensOmicsLab/PRM-script and provides a framework that can be adapted to multiple DDA/DIA data outputs and instrument-specific PRM method types.

Proteomics Defines Plasma Biomarkers for the Early Diagnosis of Biliary Atresia.

Early diagnosis of biliary atresia (BA) is crucial for improving the chances of survival and preserving the liver function of pediatric patients with BA. Herein, we performed proteomics analysis using data-independent acquisition (DIA) and parallel reaction monitoring (PRM) to explore potential biomarkers for the early diagnosis of BA compared to other non-BA jaundice cases. Consequently, we detected and validated differential protein expression in the plasma of patients with BA compared to the plasma of patients with intrahepatic cholestasis. Bioinformatics analysis revealed the enriched biological processes characteristic of BA by identifying the differential expression of specific proteins. Signaling pathway analysis revealed changes in the expression levels of proteins associated with an alteration in immunoglobulin levels, which is indicative of immune dysfunction in BA. The combination of polymeric immunoglobulin receptor expression and immunoglobulin lambda variable chain (IGL c2225_light_IGLV1-47_IGLJ2), as revealed via machine learning, provided a useful early diagnostic model for BA, with a sensitivity of 0.8, specificity of 1, accuracy of 0.89, and area under the curve value of 0.944. Thus, our study identified a possible effective plasma biomarker for the early diagnosis of BA and could help elucidate the underlying mechanisms of BA.

Automated High-Throughput Biological Sex Identification from Archeological Human Dental Enamel Using Targeted Proteomics.

Biological sex is key information for archeological and forensic studies, which can be determined by proteomics. However, the lack of a standardized approach for fast and accurate sex identification currently limits the reach of proteomics applications. Here, we introduce a streamlined mass spectrometry (MS)-based workflow for the determination of biological sex using human dental enamel. Our approach builds on a minimally invasive sampling strategy by acid etching, a rapid online liquid chromatography (LC) gradient coupled to a high-resolution parallel reaction monitoring (PRM) assay allowing for a throughput of 200 samples per day (SPD) with high quantitative performance enabling confident identification of both males and females. Additionally, we developed a streamlined data analysis pipeline and integrated it into a Shiny interface for ease of use. The method was first developed and optimized using modern teeth and then validated in an independent set of deciduous teeth of known sex. Finally, the assay was successfully applied to archeological material, enabling the analysis of over 300 individuals. We demonstrate unprecedented performance and scalability, speeding up MS analysis by 10-fold compared to conventional proteomics-based sex identification methods. This work paves the way for large-scale archeological or forensic studies enabling the investigation of entire populations rather than focusing on individual high-profile specimens. Data are available via ProteomeXchange with the identifier PXD049326.

A Framework for Quality Control in Quantitative Proteomics.

A thorough evaluation of the quality, reproducibility, and variability of bottom-up proteomics data is necessary at every stage of a workflow, from planning to analysis. We share vignettes applying adaptable quality control (QC) measures to assess sample preparation, system function, and quantitative analysis. System suitability samples are repeatedly measured longitudinally with targeted methods, and we share examples where they are used on three instrument platforms to identify severe system failures and track function over months to years. Internal QCs incorporated at the protein and peptide levels allow our team to assess sample preparation issues and to differentiate system failures from sample-specific issues. External QC samples prepared alongside our experimental samples are used to verify the consistency and quantitative potential of our results during batch correction and normalization before assessing biological phenotypes. We combine these controls with rapid analysis (Skyline), longitudinal QC metrics (AutoQC), and server-based data deposition (PanoramaWeb). We propose that this integrated approach to QC is a useful starting point for groups to facilitate rapid quality control assessment to ensure that valuable instrument time is used to collect the best quality data possible. Data are available on Panorama Public and ProteomeXchange under the identifier PXD051318.

Targeted Mass Spectrometry Analyses of Somatic Mutations in Colorectal Cancer Specimens Using Differential Ion Mobility.

Identification of K-Ras and B-Raf mutations in colorectal cancer (CRC) is essential to predict patients' response to anti-EGFR therapy and formulate appropriate therapeutic strategies to improve prognosis and survival. Here, we combined parallel reaction monitoring (PRM) with high-field asymmetric waveform ion mobility (FAIMS) to enhance mass spectrometry sensitivity and improve the identification of low-abundance K-Ras and B-Raf mutations in biological samples without immunoaffinity enrichment. In targeted LC-MS/MS analyses, FAIMS reduced the occurrence of interfering ions and enhanced precursor ion purity, resulting in a 3-fold improvement in the detection limit for K-Ras and B-Raf mutated peptides. In addition, the ion mobility separation of isomeric peptides using FAIMS facilitated the unambiguous identification of K-Ras G12D and G13D peptides. The application of targeted LC-MS/MS analyses using FAIMS is demonstrated for the detection and quantitation of B-Raf V600E, K-Ras G12D, G13D, and G12V in CRC cell lines and primary specimens.

Targeted Mass Spectrometry of Longitudinal Patient Sera Reveals LTBP1 as a Potential Surveillance Biomarker for High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSC) is the most prevalent subtype of epithelial ovarian cancer. The combination of a high rate of recurrence and novel therapies in HGSC necessitates an accurate assessment of the disease. Currently, HGSC response to treatment and recurrence are monitored via immunoassay of serum levels of the glycoprotein CA125. CA125 levels predictably rise at HGSC recurrence; however, it is likely that the disease is progressing even before it is detectable through CA125. This may explain why treating solely based on CA125 increase has not been associated with improved outcomes. Thus, additional biomarkers that monitor HGSC progression and cancer recurrence are needed. For this purpose, we developed a scheduled parallel reaction monitoring mass spectrometry (PRM-MS) assay for the quantification of four previously identified HGSC-derived glycopeptides (from proteins FGL2, LGALS3BP, LTBP1, and TIMP1). We applied the assay to quantify their longitudinal expression profiles in 212 serum samples taken from 34 HGSC patients during disease progression. Analyses revealed that LTBP1 best-mirrored tumor load, dropping as a result of cancer treatment in 31 out of 34 patients and rising at HGSC recurrence in 28 patients. Additionally, LTBP1 rose earlier during remission than CA125 in 11 out of 25 platinum-sensitive patients with an average lead time of 116.4 days, making LTBP1 a promising candidate for monitoring of HGSC recurrence.

Single Isotopologue for In-Sample Calibration and Absolute Quantitation by LC-MS/MS.

Targeted mass spectrometry (MS)-based absolute quantitative analysis has been increasingly used in biomarker discovery. The ability to accurately measure the masses by MS enabled the use of isotope-incorporated surrogates having virtually identical physiochemical properties with the target analytes as calibrators. Such a unique capacity allowed for accurate in-sample calibration. Current in-sample calibration uses multiple isotopologues or structural analogues for both the surrogate and the internal standard. Here, we simplified this common practice by using endogenous light peptides as the internal standards and used a mathematical deduction of "heavy matching light, HML" to directly quantify an endogenous analyte. This method provides all necessary assay performance parameters in the authentic matrix, including the lower limit of quantitation (LLOQ) and intercept of the calibration curve, by using only a single isotopologue of the analyte. This method can be applied to the quantitation of proteins, peptides, and small molecules. Using this method, we quantified the efficiency of heart tissue digestion and recovery using sodium deoxycholate as a detergent and two spiked exogenous proteins as mimics of heart proteins. The results demonstrated the robustness of the assay.

Multi-trait transcriptome-wide association studies with probabilistic Mendelian randomization.

A transcriptome-wide association study (TWAS) integrates data from genome-wide association studies and gene expression mapping studies for investigating the gene regulatory mechanisms underlying diseases. Existing TWAS methods are primarily univariate in nature, focusing on analyzing one outcome trait at a time. However, many complex traits are correlated with each other and share a common genetic basis. Consequently, analyzing multiple traits jointly through multivariate analysis can potentially improve the power of TWASs. Here, we develop a method, moPMR-Egger (multiple outcome probabilistic Mendelian randomization with Egger assumption), for analyzing multiple outcome traits in TWAS applications. moPMR-Egger examines one gene at a time, relies on its cis-SNPs that are in potential linkage disequilibrium with each other to serve as instrumental variables, and tests its causal effects on multiple traits jointly. A key feature of moPMR-Egger is its ability to test and control for potential horizontal pleiotropic effects from instruments, thus maximizing power while minimizing false associations for TWASs. In simulations, moPMR-Egger provides calibrated type I error control for both causal effects testing and horizontal pleiotropic effects testing and is more powerful than existing univariate TWAS approaches in detecting causal associations. We apply moPMR-Egger to analyze 11 traits from 5 trait categories in the UK Biobank. In the analysis, moPMR-Egger identified 13.15% more gene associations than univariate approaches across trait categories and revealed distinct regulatory mechanisms underlying systolic and diastolic blood pressures.

Quantitative proteomics analysis reveals the protective role of S14G-humanin in septic acute kidney injury using 4D-label-free and PRM Approaches.

Mitochondrial dysfunction contributes to septic acute kidney injury (S-AKI), making mitochondrial protection a potential therapeutic strategy. This study investigates the effects of S14G-humanin (HNG) in S-AKI, utilizing 4D-label-free and parallel reaction monitoring (PRM) techniques for proteomic analysis. An S-AKI model was created in male C57BL/6 mice using lipopolysaccharide (LPS) injection, followed by HNG administration. After 24 h, kidney tissues were analyzed for histology, biochemistry, mitochondrial function, and proteomics. HNG treatment improved renal function, reduced tubular injury, and decreased pro-inflammatory cytokines and oxidative stress markers. Proteomic analysis identified 5900 proteins, with 5111 quantifiable. HNG altered the expression of 132 proteins, with 18 selected for PRM validation. Ten of these proteins were linked to key pathways, including fatty acid degradation and PPAR signaling. This study is the first to show HNG's protective effects in S-AKI, providing insights into its mechanisms through advanced proteomic techniques.

Key Residue in the Precursor Region of M Protein Contributes to the Neurovirulence and Neuroinvasiveness of the African Lineage of Zika Virus.

The Zika virus (ZIKV) represents an important global health threat due to its unusual association with congenital Zika syndrome. ZIKV strains are phylogenetically grouped into the African and Asian lineages. However, the viral determinants underlying the phenotypic differences between the lineages remain unknown. Here, multiple sequence alignment revealed a highly conserved residue at position 21 of the premembrane (prM) protein, which is glutamic acid and lysine in the Asian and African lineages, respectively. Using reverse genetics, we generated a recombinant virus carrying an E21K mutation based on the genomic backbone of the Asian lineage strain FSS13025 (termed E21K). The E21K mutation significantly increased viral replication in multiple neural cell lines with a higher ratio of M to prM production. Animal studies showed E21K exhibited increased neurovirulence in suckling mice, leading to more severe defects in mouse brains by causing more neural cell death and destruction of hippocampus integrity. Moreover, the E21K substitution enhanced neuroinvasiveness in interferon alpha/beta (IFN-α/ß) receptor knockout mice, as indicated by the increased mortality, and enhanced replication in mouse brains. The global transcriptional analysis showed E21K infection profoundly altered neuron development networks and induced stronger antiviral immune response than wild type (WT) in both neural cells and mouse brains. More importantly, the reverse K21E mutation based on the genomic backbone of the African strain MR766 caused less mouse neurovirulence. Overall, our findings support the 21st residue of prM functions as a determinant for neurovirulence and neuroinvasiveness of the African lineage of ZIKV. IMPORTANCE The suspected link of Zika virus (ZIKV) to birth defects led the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. ZIKV has been identified to have two dominant phylogenetic lineages, African and Asian. Significant differences exist between the two lineages in terms of neurovirulence and neuroinvasiveness in mice. However, the viral determinants underlying the phenotypic differences are still unknown. Here, combining reverse genetics, animal studies, and global transcriptional analysis, we provide evidence that a single E21K mutation of prM confers to the Asian lineage strain FSS130125 significantly enhanced replication in neural cell lines and more neurovirulent and neuroinvasiveness phenotypes in mice. Our findings support that the highly conserved residue at position 21 of prM functions as a determinant of neurovirulence and neuroinvasiveness of the African lineage of ZIKV in mice.

Simultaneous targeted and discovery-driven clinical proteotyping using hybrid-PRM/DIA.

BACKGROUND: Clinical samples are irreplaceable, and their transformation into searchable and reusable digital biobanks is critical for conducting statistically empowered retrospective and integrative research studies. Currently, mainly data-independent acquisition strategies are employed to digitize clinical sample cohorts comprehensively. However, the sensitivity of DIA is limited, which is why selected marker candidates are often additionally measured targeted by parallel reaction monitoring. METHODS: Here, we applied the recently co-developed hybrid-PRM/DIA technology as a new intelligent data acquisition strategy that allows for the comprehensive digitization of rare clinical samples at the proteotype level. Hybrid-PRM/DIA enables enhanced measurement sensitivity for a specific set of analytes of current clinical interest by the intelligent triggering of multiplexed parallel reaction monitoring (MSxPRM) in combination with the discovery-driven digitization of the clinical biospecimen using DIA. Heavy-labeled reference peptides were utilized as triggers for MSxPRM and monitoring of endogenous peptides. RESULTS: We first evaluated hybrid-PRM/DIA in a clinical context on a pool of 185 selected proteotypic peptides for tumor-associated antigens derived from 64 annotated human protein groups. We demonstrated improved reproducibility and sensitivity for the detection of endogenous peptides, even at lower concentrations near the detection limit. Up to 179 MSxPRM scans were shown not to affect the overall DIA performance. Next, we applied hybrid-PRM/DIA for the integrated digitization of biobanked melanoma samples using a set of 30 AQUA peptides against 28 biomarker candidates with relevance in molecular tumor board evaluations of melanoma patients. Within the DIA-detected approximately 6500 protein groups, the selected marker candidates such as UFO, CDK4, NF1, and PMEL could be monitored consistently and quantitatively using MSxPRM scans, providing additional confidence for supporting future clinical decision-making. CONCLUSIONS: Combining PRM and DIA measurements provides a new strategy for the sensitive and reproducible detection of protein markers from patients currently being discussed in molecular tumor boards in combination with the opportunity to discover new biomarker candidates.

Plasma proteomic characterization of colorectal cancer patients with FOLFOX chemotherapy by integrated proteomics technology.

BACKGROUND: Colorectal Cancer (CRC) is a prevalent form of cancer, and the effectiveness of the main postoperative chemotherapy treatment, FOLFOX, varies among patients. In this study, we aimed to identify potential biomarkers for predicting the prognosis of CRC patients treated with FOLFOX through plasma proteomic characterization. METHODS: Using a fully integrated sample preparation technology SISPROT-based proteomics workflow, we achieved deep proteome coverage and trained a machine learning model from a discovery cohort of 90 CRC patients to differentiate FOLFOX-sensitive and FOLFOX-resistant patients. The model was then validated by targeted proteomics on an independent test cohort of 26 patients. RESULTS: We achieved deep proteome coverage of 831 protein groups in total and 536 protein groups in average for non-depleted plasma from CRC patients by using a Orbitrap Exploris 240 with moderate sensitivity. Our results revealed distinct molecular changes in FOLFOX-sensitive and FOLFOX-resistant patients. We confidently identified known prognostic biomarkers for colorectal cancer, such as S100A4, LGALS1, and FABP5. The classifier based on the biomarker panel demonstrated a promised AUC value of 0.908 with 93% accuracy. Additionally, we established a protein panel to predict FOLFOX effectiveness, and several proteins within the panel were validated using targeted proteomic methods. CONCLUSIONS: Our study sheds light on the pathways affected in CRC patients treated with FOLFOX chemotherapy and identifies potential biomarkers that could be valuable for prognosis prediction. Our findings showed the potential of mass spectrometry-based proteomics and machine learning as an unbiased and systematic approach for discovering biomarkers in CRC.

Absolute quantitation of human wild-type DNAI1 protein in lung tissue using a nanoLC-PRM-MS-based targeted proteomics approach coupled with immunoprecipitation.

BACKGROUND: Dynein axonemal intermediate chain 1 protein (DNAI1) plays an essential role in cilia structure and function, while its mutations lead to primary ciliary dyskinesia (PCD). Accurate quantitation of DNAI1 in lung tissue is crucial for comprehensive understanding of its involvement in PCD, as well as for developing the potential PCD therapies. However, the current protein quantitation method is not sensitive enough to detect the endogenous level of DNAI1 in complex biological matrix such as lung tissue. METHODS: In this study, a quantitative method combining immunoprecipitation with nanoLC-MS/MS was developed to measure the expression level of human wild-type (WT) DNAI1 protein in lung tissue. To our understanding, it is the first immunoprecipitation (IP)-MS based method for absolute quantitation of DNAI1 protein in lung tissue. The DNAI1 quantitation was achieved through constructing a standard curve with recombinant human WT DNAI1 protein spiked into lung tissue matrix. RESULTS: This method was qualified with high sensitivity and accuracy. The lower limit of quantitation of human DNAI1 was 4 pg/mg tissue. This assay was successfully applied to determine the endogenous level of WT DNAI1 in human lung tissue. CONCLUSIONS: The results clearly demonstrate that the developed assay can accurately quantitate low-abundance WT DNAI1 protein in human lung tissue with high sensitivity, indicating its high potential use in the drug development for DNAI1 mutation-caused PCD therapy.