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Single-cell proteomics (SCP) aims to characterize the proteome of individual cells, providing insights into complex biological systems. It reveals subtle differences in distinct cellular populations that bulk proteome analysis may overlook, which is essential for understanding disease mechanisms and developing targeted therapies. Mass spectrometry (MS) methods in SCP allow the identification and quantification of thousands of proteins from individual cells. Two major challenges in SCP are the limited material in single-cell samples necessitating highly sensitive analytical techniques and the efficient processing of samples, as each biological sample requires thousands of single cell measurements. This review discusses MS advancements to mitigate these challenges using data-dependent acquisition (DDA) and data-independent acquisition (DIA). Additionally, we examine the use of short liquid chromatography gradients and sample multiplexing methods that increase the sample throughput and scalability of SCP experiments. We believe these methods will pave the way for improving our understanding of cellular heterogeneity and its implications for systems biology.
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BACKGROUND: Increasing epidemiologic studies have shown a positive correlation between obesity and chronic diarrhea. Nevertheless, the precise etiology remains uncertain. METHODS: We performed a comprehensive proteomics analysis utilizing the data-independent acquisition (DIA) technique on jejunal tissues from patients with obesity and chronic diarrhea (OD, n = 33), obese patients (OB, n = 10), and healthy controls (n = 8). Differentially expressed proteins (DEPs) in OD vs. control and OD vs. OB comparisons were subjected to pathway enrichment and protein-protein interaction (PPI) network analysis. Machine learning algorithms were adopted on overlapping DEPs in both comparisons. The candidate protein was further validated using Western blot, immunohistochemistry (IHC), and in vitro experiments. RESULTS: We identified 189 and 228 DEPs in OD vs. control and OD vs. OB comparisons, respectively. DEPs in both comparisons were co-enriched in extracellular matrix (ECM) organization. Downregulated DEPs were associated with tight junction and ECM-receptor interaction in OD vs. control and OD vs. OB comparisons, respectively. Machine learning algorithms selected 3 proteins from 14 overlapping DEPs in both comparisons, among which collagen alpha-1(III) chain (COL3A1) was identified as a core protein in PPI networks. Western blot and IHC verified the expression of COL3A1. Moreover, the tight junction-related proteins decreased after the knockdown of COL3A1 in Caco2 intestinal cells upon PA challenge, consistent with the proteomics results. CONCLUSIONS: We generated in-depth profiling of a proteomic dataset from samples of OD patients and provided unique insights into disease pathogenesis. COL3A1 was involved in the crosstalk between obesity and intestinal homeostasis via the ECM-receptor interaction pathway.
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BACKGROUND: Despite extensive research, the identification of effective biomarkers for early prediction of preterm birth (PTB) continues to be a challenging endeavor. This study aims to identify amniotic fluid (AF) protein biomarkers useful for the early diagnosis of PTB. METHODS: We initially identified the protein expression profiles in the AF of women with PTB (n = 22) and full-term birth (FTB, n = 22), from the First People's Hospital of Yunnan Province who underwent amniocentesis from November 2019 to February 2020, using mass spectrometry employing the data-independent acquisition (DIA) technique, and then analyzed differentially expressed proteins (DEPs). Subsequently, the least absolute shrinkage and selection operator (LASSO) and random forest analysis were employed to further screen the key proteins for PTB biomarker identification. The receiver operating characteristic (ROC) analysis, calibration plots, and decision curve analyses (DCA) were utilized to assess the discrimination and calibration of the key biomarkers. RESULTS: A total of 25 DEPs were identified between the PTB and FTB groups, comprising 13 up-regulated and 12 down-regulated proteins. Three key protein biomarkers for early PTB diagnosis were identified: IL1RL1 (interleukin-1 receptor-like 1), APOE (apolipoprotein E), and NECTIN4 (nectin cell adhesion molecule 4). The results of the ROC analysis showed that the area under the curve (AUC) of the three proteins combined as a biomarker for early diagnosis of PTB was 0.913 (95% CI: 0.823-1.000), with a sensitivity of 0.864 and a specificity of 0.955, both superior to those of the individual biomarkers. Bootstrap internal validation revealed a concordance index (C-index) of 0.878, with a sensitivity of 0.812 and a specificity of 0.773, indicating the robust predictive performance of these biomarkers. CONCLUSIONS: We identified three previously unexplored yet potentially useful protein biomarkers in AF for early PTB diagnosis: IL1RL1, APOE, and NECTIN4.
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Líquido Amniótico , Apolipoproteínas E , Biomarcadores , Nascimento Prematuro , Proteômica , Humanos , Feminino , Nascimento Prematuro/diagnóstico , Nascimento Prematuro/metabolismo , Gravidez , Adulto , Biomarcadores/metabolismo , Biomarcadores/análise , Proteômica/métodos , Líquido Amniótico/metabolismo , Líquido Amniótico/química , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/metabolismo , Nectinas/metabolismo , Curva ROC , AmniocenteseRESUMO
PURPOSE: Behçet's disease-associated uveitis (BDU) is a severe, recurrent inflammatory condition affecting the eye and is part of a systemic vasculitis with unknown etiology, making biomarker discovery essential for disease management. In this study, we intend to investigate potential urinary biomarkers to monitor the disease activity of BDU. METHODS: Firstly, label-free data-dependent acquisition (DDA) and tandem mass tag (TMT)-labeled quantitative proteomics methods were used to profile the proteomes of urine from active and quiescent BDU patients, respectively. For further exploration, the remaining fifty urine samples were analyzed by a data-independent acquisition (DIA) quantitative proteomics method. RESULTS: Twenty-nine and 21 differential proteins were identified in the same urine from BDU patients by label-free DDA and TMT-labeled analyses, respectively. Seventy-nine differentially expressed proteins (DEPs) were significantly changed in other active BDU urine samples compared to those in quiescent BDU urine samples by IDA analysis. Gene Ontology (GO) and protein-protein interaction (PPI) analyses revealed that the DEPs were associated with multiple functions, including the immune and neutrophil activation responses. Finally, seven proteins were identified as candidate biomarkers for BDU monitoring and recurrence prediction, namely, CD38, KCRB, DPP4, FUCA2, MTPN, S100A8 and S100A9. CONCLUSIONS: Our results showed that urine can be a good source of biomarkers for BDU. These dysregulated proteins provide potential urinary biomarkers for BDU activity monitoring and provide valuable clues for the analysis of the pathogenic mechanisms of BDU.
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Síndrome de Behçet , Biomarcadores , Proteoma , Proteômica , Uveíte , Humanos , Síndrome de Behçet/urina , Síndrome de Behçet/diagnóstico , Síndrome de Behçet/metabolismo , Biomarcadores/urina , Masculino , Feminino , Uveíte/urina , Uveíte/diagnóstico , Uveíte/metabolismo , Proteoma/análise , Proteoma/metabolismo , Adulto , Proteômica/métodos , Pessoa de Meia-Idade , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: Identification of suitable biomarkers that facilitate the screening and evaluation of pediatric obstructive sleep apnea (OSA) and its severity was explored. METHODS: Data-independent acquisition quantitative proteomic analysis was employed to identify serum and urine proteins with differential expression patterns between children with OSA and controls. Differentially expressed proteins that gradually increased or decreased with the severity of OSA were retained as potential biomarkers and underwent ELISA validation. RESULTS: We found that with increasing severity of OSA, there was a gradual upregulation of 34 proteins in the serum and 124 proteins in the urine, along with a respective downregulation of 10 serum proteins and 64 urinary proteins in the initial cohort of 40 children. These proteins primarily participate in immune activation, the complement pathway, oxygen transport, and reactive oxygen metabolism. Notably, cathepsin Z exhibited a positive correlation with the obstructive apnea hypopnea index, whereas sex hormone-binding globulin (SHBG) was negatively correlated. These proteins were then validated by ELISA in an independent cohort (n=21). Circulating cathepsin Z and SHBG levels displayed acceptable diagnostic performance of OSA with AUC values of 0.863 and 0.738, respectively. CONCLUSIONS: We identified two promising circulating proteins as novel biomarkers for clinical diagnosis and assessment of pediatric OSA severity. Furthermore, the comprehensive proteomic profile in pediatric OSA should aid in exploring the underlying pathophysiological mechanisms associated with this prevalent condition.
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Legumes perform symbiotic nitrogen fixation through rhizobial bacteroids housed in specialised root nodules. The biochemical process is energy-intensive and consumes a huge carbon source to generate sufficient reducing power. To maintain the symbiosis, malate is supplied by legume nodules to bacteroids as their major carbon and energy source in return for ammonium ions and nitrogenous compounds. To sustain the carbon supply to bacteroids, nodule cells undergo drastic reorganisation of carbon metabolism. Here, a comprehensive quantitative comparison of the mitochondrial proteomes between root nodules and uninoculated roots was performed using data-independent acquisition proteomics, revealing the modulations in nodule mitochondrial proteins and pathways in response to carbon reallocation. Corroborated our findings with that from the literature, we believe nodules preferably allocate cytosolic phosphoenolpyruvates towards malate synthesis in lieu of pyruvate synthesis, and nodule mitochondria prefer malate over pyruvate as the primary source of NADH for ATP production. Moreover, the differential regulation of respiratory chain-associated proteins suggests that nodule mitochondria could enhance the efficiencies of complexes I and IV for ATP synthesis. This study highlighted a quantitative proteomic view of the mitochondrial adaptation in soybean nodules.
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Colorectal cancer (CRC) is projected to become the third most diagnosed and third most fatal cancer in the United States by 2024, with early onset CRC on the rise. Research is constantly underway to discover novel therapeutics for the treatment of various cancers to improve patient outcomes and survival. Fatty acid synthase (FAS) has become a druggable target of interest for the treatment of many different cancers. One such inhibitor, TVB-2640, has gained popularity for its high specificity for FAS and has entered a phase 1 clinical trial for the treatment of solid tumors. However, the distinct molecular differences that occur upon inhibition of FAS have yet to be understood. Here, we conduct proteomics and phosphoproteomics analyses on HCT 116 and HT-29 CRC spheroids inhibited with either a generation 1 (cerulenin) or generation 2 (TVB-2640) FAS inhibitor. Proteins involved in lipid metabolism and cellular respiration were altered in abundance. It was also observed that proteins involved in ferroptosisâan iron mediated form of cell deathâwere altered. These results show that HT-29 spheroids exposed to cerulenin or TVB-2640 are undergoing a ferroptotic death mechanism. The data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the identifier PXD050987.
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Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized the SWATH-DIA method using a narrow isolation window placement approach, improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios ofEscherichia coli, yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.
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Proteômica , Proteômica/métodos , Humanos , Animais , Camundongos , Células HEK293 , Fígado/metabolismo , Fígado/química , Peptídeos/química , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteoma/análise , Escherichia coli/metabolismo , Escherichia coli/genética , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas/métodosRESUMO
In order to assess homeostatic mechanisms in the lung after COVID-19, changes in the protein signature of bronchoalveolar lavage from 45 patients with mild to moderate disease at three phases (acute, recovery, and convalescent) are evaluated over a year. During the acute phase, inflamed and uninflamed phenotypes are characterized by the expression of tissue repair and host defense response molecules. With recovery, inflammatory and fibrogenic mediators decline and clinical symptoms abate. However, at 9 months, quantified radiographic abnormalities resolve in the majority of patients, and yet compared to healthy persons, all showed ongoing activation of cellular repair processes and depression of the renin-kallikrein-kinin, coagulation, and complement systems. This dissociation of prolonged reparative processes from symptom and radiographic resolution suggests that occult ongoing disruption of the lung proteome is underrecognized and may be relevant to recovery from other serious viral pneumonias.
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COVID-19 , Pulmão , Proteoma , SARS-CoV-2 , Humanos , COVID-19/metabolismo , COVID-19/patologia , COVID-19/virologia , Proteoma/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Pulmão/diagnóstico por imagem , Feminino , Masculino , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , Estudos Longitudinais , Adulto , Líquido da Lavagem Broncoalveolar/química , IdosoRESUMO
Mass spectrometry is broadly employed to study complex molecular mechanisms in various biological and environmental fields, enabling 'omics' research such as proteomics, metabolomics, and lipidomics. As study cohorts grow larger and more complex with dozens to hundreds of samples, the need for robust quality control (QC) measures through automated software tools becomes paramount to ensure the integrity, high quality, and validity of scientific conclusions from downstream analyses and minimize the waste of resources. Since existing QC tools are mostly dedicated to proteomics, automated solutions supporting metabolomics are needed. To address this need, we developed the software PeakQC, a tool for automated QC of MS data that is independent of omics molecular types (i.e., omics-agnostic). It allows automated extraction and inspection of peak metrics of precursor ions (e.g., errors in mass, retention time, arrival time) and supports various instrumentations and acquisition types, from infusion experiments or using liquid chromatography and/or ion mobility spectrometry front-end separations and with/without fragmentation spectra from data-dependent or independent acquisition analyses. Diagnostic plots for fragmentation spectra are also generated. Here, we describe and illustrate PeakQC's functionalities using different representative data sets, demonstrating its utility as a valuable tool for enhancing the quality and reliability of omics mass spectrometry analyses.
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BACKGROUND: Protein phosphorylation by protein kinases plays a pivotal role in increasing protein diversity, thereby influencing various cellular functions. However, due to the relatively low abundance of phosphopeptides in a mixture of peptides and the ion-suppression effect of non-phosphorylated peptides, the detection of phosphopeptides is not straightforward. RESULTS: Herein, a quantitative high-throughput platform was developed for assessing multikinase activity using nano-LC-MS/MS with a data-independent acquisition (DIA) approach. This platform was evaluated by studying the kinase activity in Taxol-treated SKOV3 cells. A library containing 38 peptide substrates was designed and analyzed to determine the activities of major kinases involved in cancer development. Twenty-three synthetic peptide substrates showed significant phosphorylation changes in triplicate biological experiments, as further verified by western blotting. Our findings reveal that Taxol suppressed SKOV3 cell survival by activating AMPK and suppressing the PI3K-Akt-dependent pathway, ultimately leading to mTOR inhibition. Furthermore, in combination with ERK, Akt, SGK, CK1, and ErbB2 inhibitors, Taxol enhanced the inhibitory effect on ovarian cancer. SIGNIFICANCE: This platform can be an attractive approach for large-scale kinase activity studies to comprehensively uncover the mechanisms of drug-disease treatment and to investigate a more effective therapy strategy.
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Neoplasias Ovarianas , Paclitaxel , Espectrometria de Massas em Tandem , Paclitaxel/farmacologia , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Feminino , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/química , Sobrevivência Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Nanotecnologia , Ensaios de Seleção de Medicamentos Antitumorais , Proteínas Quinases/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
Ion mobility (IM) is often combined with LC-MS experiments to provide an additional dimension of separation for complex sample analysis. While highly complex samples are better characterized by the full dimensionality of LC-IM-MS experiments to uncover new information, downstream data analysis workflows are often not equipped to properly mine the additional IM dimension. For many samples the data acquisition benefits of including IM separations are all that is necessary to uncover sample information and the full dimensionality of the data is not required for data analysis. Postacquisition reduction and adaptation of the dimensions of LC-IM-MS and IM-MS experiments into an LC-MS format opens the possibility to use a plethora of existing software tools. In this work, we developed data file conversion tools to reduce the complexity of IM data analysis. Three data file transformations are introduced in the PNNL PreProcessor software: (1) mapping the IM axis to the LC axis for IM-MS data, (2) converting the drift time vs m/z space to CCS/z vs m/z space, and (3) transforming All Ions IM/MS mobility aligned fragmentation data to a standard LC-MS DDA data file format. These new data file conversions are demonstrated with corresponding lipidomics and proteomics workflows that leverage existing LC-MS data analysis software to highlight the benefits of the data transformations.
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Aberrant glycosylation has gained significant interest for biomarker discovery. However, low detectability, complex glycan structures, and heterogeneity present challenges in glycoprotein assay development. Using haptoglobin (Hp) as a model, we developed an integrated platform combining functionalized magnetic nanoparticles and zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) for highly specific glycopeptide enrichment, followed by a data-independent acquisition (DIA) strategy to establish a deep cancer-specific Hp-glycosylation profile in hepatitis B virus (HBV, n = 5) and hepatocellular carcinoma (HCC, n = 5) patients. The DIA strategy established one of the deepest Hp-glycosylation landscapes (1029 glycopeptides, 130 glycans) across serum samples, including 54 glycopeptides exclusively detected in HCC patients. Additionally, single-shot DIA searches against a DIA-based spectral library outperformed the DDA approach by 2-3-fold glycopeptide coverage across patients. Among the four N-glycan sites on Hp (N-184, N-207, N-211, N-241), the total glycan type distribution revealed significantly enhanced detection of combined fucosylated-sialylated glycans, which were the most dominant glycoforms identified in HCC patients. Quantitation analysis revealed 48 glycopeptides significantly enriched in HCC (p < 0.05), including a hybrid monosialylated triantennary glycopeptide on the N-184 site with nearly none-to-all elevation to differentiate HCC from the HBV group (HCC/HBV ratio: 2462 ± 766, p < 0.05). In summary, DIA-MS presents an unbiased and comprehensive alternative for targeted glycoproteomics to guide discovery and validation of glyco-biomarkers.
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Carcinoma Hepatocelular , Glicopeptídeos , Haptoglobinas , Neoplasias Hepáticas , Polissacarídeos , Humanos , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/metabolismo , Glicosilação , Haptoglobinas/metabolismo , Haptoglobinas/análise , Haptoglobinas/química , Polissacarídeos/sangue , Polissacarídeos/química , Polissacarídeos/análise , Glicopeptídeos/sangue , Glicopeptídeos/análise , Glicopeptídeos/química , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Biomarcadores Tumorais/sangue , Hepatite B/virologia , Hepatite B/sangue , Vírus da Hepatite B/química , Interações Hidrofóbicas e HidrofílicasRESUMO
Compared to advancements in single-cell proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We present a simple and rapid one-pot phosphoproteomics workflow (SOP-Phos) integrated with data-independent acquisition mass spectrometry (DIA-MS) for microscale phosphoproteomic analysis. SOP-Phos adapts sodium deoxycholate based one-step lysis, reduction/alkylation, direct trypsinization, and phosphopeptide enrichment by TiO2 beads in a single-tube format. By reducing surface adsorptive losses via utilizing n-dodecyl ß-d-maltoside precoated tubes and shortening the digestion time, SOP-Phos is completed within 3-4 h with a 1.4-fold higher identification coverage. SOP-Phos coupled with DIA demonstrated >90% specificity, enhanced sensitivity, lower missing values (<1%), and improved reproducibility (8%-10% CV). With a sample size-comparable spectral library, SOP-Phos-DIA identified 33,787 ± 670 to 22,070 ± 861 phosphopeptides from 5 to 0.5 µg cell lysate and 30,433 ± 284 to 6,548 ± 21 phosphopeptides from 50,000 to 2,500 cells. Such sensitivity enabled mapping key lung cancer signaling sites, such as EGFR autophosphorylation sites Y1197/Y1172 and drug targets. The feasibility of SOP-Phos-DIA was demonstrated on EGFR-TKI sensitive and resistant cells, revealing the interplay of multipathway Hippo-EGFR-ERBB signaling cascades underlying the mechanistic insight into EGFR-TKI resistance. Overall, SOP-Phos-DIA is an efficient and robust protocol that can be easily adapted in the community for microscale phosphoproteomic analysis.
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Fosfopeptídeos , Fosfoproteínas , Proteômica , Fluxo de Trabalho , Proteômica/métodos , Humanos , Fosfopeptídeos/análise , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/química , Reprodutibilidade dos Testes , Receptores ErbB/metabolismo , Linhagem Celular Tumoral , Fosforilação , Titânio/química , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas/métodosRESUMO
Fragment correlation mass spectrometry correlates ion pairs generated from the same fragmentation pathway, achieved by covariance mapping of tandem mass spectra generated with an unmodified linear ion trap without preseparation. We enable the identification of different precursors at different charge states in a complex mixture from a large isolation window, empowering an analytical approach for data-independent acquisition. The method resolves and matches isobaric fragments, internal ions, and disulfide bond fragments. We suggest that this method represents a major advance for analyzing structures of biopolymers in mixtures.
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Biofluids such as blood plasma are rich reservoirs of potential biomarkers for disease diagnosis, prognosis, and prediction of treatment response. However, mass spectrometry analysis of circulating plasma proteins remains challenging. The introduction of data-independent acquisition mass spectrometry (DIA-MS) is an important step toward addressing detection of less abundant plasma proteins. Numerous plasma peptide MS/MS spectral library datasets produced from extensive plasma fractionation are accessible from public archives, and these can be repurposed as spectral reference libraries to increase the depth of proteomic analysis when DIA-MS is used. Here we describe the workflow that relies on reusing the existing spectral reference libraries by populating them with locally obtained peptide MS/MS data acquired by DIA-MS. This approach was demonstrated effectively to identify putative plasma biomarkers of response to neoadjuvant chemotherapy in the setting of pancreatic ductal adenocarcinoma (PDAC) (O'Rourke et al., J Proteomics 231:103998, 2021).
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Biomarcadores Tumorais , Neoplasias Pancreáticas , Proteômica , Espectrometria de Massas em Tandem , Humanos , Biomarcadores Tumorais/sangue , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/sangue , Biblioteca de Peptídeos , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/tratamento farmacológico , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Terapia Neoadjuvante/métodosRESUMO
OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.
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Schizophrenia is a severe psychological disorder. The current diagnosis mainly relies on clinical symptoms and lacks laboratory evidence, which makes it very difficult to make an accurate diagnosis especially at an early stage. Plasma protein profiles of schizophrenia patients were obtained and compared with healthy controls using 4D-DIA proteomics technology. Furthermore, 79 DEPs were identified between schizophrenia and healthy controls. GO functional analysis indicated that DEPs were predominantly associated with responses to toxic substances and platelet aggregation, suggesting the presence of metabolic and immune dysregulation in patients with schizophrenia. KEGG pathway enrichment analysis revealed that DEPs were primarily enriched in the chemokine signaling pathway and cytokine receptor interactions. A diagnostic model was ultimately established, comprising three proteins, namely, PFN1, GAPDH and ACTBL2. This model demonstrated an AUC value of 0.972, indicating its effectiveness in accurately identifying schizophrenia. PFN1, GAPDH and ACTBL2 exhibit potential as biomarkers for the early detection of schizophrenia. The findings of our studies provide novel insights into the laboratory-based diagnosis of schizophrenia.
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Biomarcadores , Profilinas , Proteômica , Esquizofrenia , Esquizofrenia/metabolismo , Esquizofrenia/diagnóstico , Esquizofrenia/sangue , Humanos , Biomarcadores/sangue , Biomarcadores/metabolismo , Proteômica/métodos , Profilinas/metabolismo , Feminino , Masculino , Adulto , Estudos de Casos e Controles , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Pessoa de Meia-Idade , Proteínas Sanguíneas/análise , Proteoma/análiseRESUMO
Clinical and biological samples are often scarce and precious (e.g., rare cell isolates, microneedle tissue biopsies, small-volume liquid biopsies, and even single cells or organelles). Typical large-scale proteomic methods, where significantly higher protein amounts are analyzed, are not directly transferable to the analysis of limited samples due to their incompatibility with pg-, ng-, and low-µg-level protein sample amounts. Here, we report the on-microsolid-phase extraction tip (OmSET)-based sample preparation workflow for sensitive analysis of limited biological samples to address this challenge. The developed platform was successfully tested for the analysis of 100-10,000 typical mammalian cells and is scalable to allow for lower and larger protein amounts and more samples to be analyzed (i.e., higher throughput of analysis).
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Proteômica , Extração em Fase Sólida , Fluxo de Trabalho , Proteômica/métodos , Humanos , Extração em Fase Sólida/métodos , Proteínas/análise , Proteoma/análiseRESUMO
INTRODUCTION: Compound annotation is always a challenging step in metabolomics studies. The molecular networking strategy has been developed recently to organize the relationship between compounds as a network based on their tandem mass (MS2) spectra similarity, which can be used to improve compound annotation in metabolomics analysis. OBJECTIVE: This study used Bupleuri Radix from different geographic areas to evaluate the performance of molecular networking strategy for compound annotation in liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. METHODOLOGY: The Bupleuri Radix extract was analyzed by LC-quadrupole time-of-flight MS under MSe acquisition mode. After raw data preprocessing, the resulting dataset was used for statistical analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The chemical makers related to the sample growth place were selected using variable importance in projection (VIP) > 2, fold change (FC) > 2, and p < 0.05. The molecular networking analysis was applied to conduct the compound annotation. RESULTS: The score plots of PCA showed that the samples were classified into two clusters depending on their growth place. Then, the PLS-DA model was constructed to explore the chemical changes of the samples further. Sixteen compounds were selected as chemical makers and tentatively annotated by the feature-based molecular networking (FBMN) analysis. CONCLUSION: The results showed that the molecular networking method fully exploits the MS information and is a promising tool for facilitating compound annotation in metabolomics studies. However, the software used for feature extraction influenced the results of library searching and molecular network construction, which need to be taken into account in future studies.