High-throughput Proteomic Profiling Research Articles

Targeted proteomics analysis of plasma proteins using recombinant protein standards for addition only workflows.

Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation<10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

High throughput proteomic and metabolic profiling identified target correction of metabolic abnormalities as a novel therapeutic approach in head and neck paraganglioma

Head and neck paragangliomas (HNPGLs) are rare neoplasms that represent difficult treatment paradigms in neurotology. Germline mutations in genes encoding succinate dehydrogenase (SDH) are the cause of nearly all familial HNPGLs. However, the molecular mechanisms underlying tumorigenesis remain unclear. Mutational analysis identified 6 out of 14 HNPGLs harboring clinicopathologic SDH gene mutations. The SDHB gene was most frequently mutated in these patients, and western blot showed loss of SDHB protein in tumors with SDHB mutations. The paraganglioma cell line (PGL-626) was established from a sample that harbored a missense SDHB mutation (c.649C > T). Spectrometric analysis using tandem mass tags identified 151 proteins significantly differentially expressed in HNPGLs compared with normal nerves. Bioinformatics analyses confirmed the high level of enrichment of oxidative phosphorylation and metabolism pathways in HNPGLs. The mitochondrial complex subunits NDUFA2, NDUFA10, and NDUFA4, showed the most significantly increased expression and were localized predominantly in the cytoplasm of PGL-626 cells. The mitochondrial complex I inhibitor metformin exerted dose-dependent inhibitory effects on PGL-626 cells via cooperative down-regulation of NDUFA2, 4, and 10, with a significant decrease in the levels of reactive oxygen species and mitochondrial membrane potential. Further metabolomic analysis of PGL-626 cells showed that metabolites involved in central carbon metabolism in cancer and sphingolipid signaling pathways, pantothenate and CoA biosynthesis, and tryptophan and carbon metabolism were significantly altered after metformin treatment. Thus, this study provides insights into the molecular mechanisms underlying HNPGL tumorigenesis and identifies target correction of metabolic abnormalities as a novel therapeutic approach for this disease.

High-Throughput Proteomic Profiling of Nipple Aspirate Fluid from Breast Cancer Patients Compared with Non-Cancer Controls: A Step Closer to Clinical Feasibility.

Background: Early detection of breast cancer (BC) is critical for increasing survival rates. However, current imaging approaches can provide ambiguous results, requiring invasive tissue biopsy for a definitive diagnosis. Multi-dimensional mass spectrometric analysis has highlighted the invaluable potential of nipple aspirate fluid (NAF) as a non-invasive source of early detection biomarkers, by identifying a multitude of proteins representative of the changing breast microenvironment. However, technical challenges with biomarker validation in large cohorts remain due to low sample throughput, impeding progress towards clinical utility. Rather, by employing a high-throughput method, that is more practicable for clinical utility, perturbations of the most abundant NAF proteins in BC patients compared with non-cancer (NC) controls could be monitored and validated in larger groups. Method: We characterized matched NAF pairs from BC (n = 9) and NC (n = 4) volunteers, using a rapid one dimensional liquid chromatography-mass spectrometry (1D LC-MS/MS) approach. Results: Overall, 198 proteins were relatively quantified, of which 40 were significantly differentiated in BC samples, compared with NC (p ≤ 0.05), with 26 upregulated and 14 downregulated. An imbalance in immune response and proteins regulating cell growth, maintenance and communication were identified. Conclusions: Our findings show 1D LC-MS/MS can quantify changes reflected in the NAF proteome associated with breast cancer development.

High-throughput proteomic profiling reveals mechanisms of action of AMG925, a dual FLT3-CDK4/6 kinase inhibitor targeting AML and AML stem/progenitor cells.

FLT3 mutations, which are found in a third of patients with acute myeloid leukemia (AML), are associated with poor prognosis. Responses to currently available FLT3 inhibitors in AML patients are typically transient and followed by disease recurrence. Thus, FLT3 inhibitors with new inhibitory mechanisms are needed to improve therapeutic outcomes. AMG925 is a novel, potent, small-molecule dual inhibitor of FLT3 and CDK4/6. In this study. we determined the antileukemic effects and mechanisms of action of AMG925 in AML cell lines and primary samples, in particular AML stem/progenitor cells. AMG925 inhibited cell growth and promoted apoptosis in AML cells with or without FLT3 mutations. Reverse-phase protein array profiling confirmed its on-target effects on FLT3-CDK4/6-regulated pathways and identified unrevealed signaling network alterations in AML blasts and stem/progenitor cells in response to AMG925. Mass cytometry identified pathways that may confer resistance to AMG925 in phenotypically defined AML stem/progenitor cells and demonstrated that combined blockade of FLT3-CDK4/6 and AKT/mTOR signaling facilitated stem cell death. Our findings provide a rationale for the mechanism-based inhibition of FLT3-CDK4/6 and for combinatorial approaches to improve the efficacy of FLT3 inhibition in both FLT3 wild-type and FLT3-mutated AML.

Proteomic profiling in extracellular vesicles for cancer detection and monitoring.

Extracellular vesicles (EVs) are nanometer-size lipid vesicles released by cells, which play essential biological functions in intercellular communication. Increasing evidence indicates that EVs participate in cancer development, including invasion, migration, metastasis, and cancer immune modulation. One of the key mechanisms is that EVs affect different cells in the tumor microenvironment through surface-anchor proteins and protein cargos. Moreover, proteins specifically expressed in tumor-derived EVs can be applied in cancer diagnosis and monitoring. Besides, the EV proteome also helps to understand drug resistance in cancers and to guide clinical medication. With the development of mass spectrometry and array-based multi-protein detection, the research of EV proteomics has entered a new era. The high-throughput parallel proteomic profiling based on these new platforms allows us to study the impact of EV proteome on cancer progression more comprehensively and to describe the proteomic landscape in cancers with more details. In this article, we review the role and function of different types of EVs in cancer progression. More importantly, we summarize the proteomic profiling of EVs based on different methods and the application of EV proteome in cancer detection and monitoring.

Establishment of a New-generation High-throughput Proteomic Profiling of Transcription Factor in Human Atrial Tissue

To explore for the establishment of an experimental technique for profiling transcription factors, namely transcription factor response elements (TFRE), with high throughput and efficiency using human atrial tissue. Postoperative right atrial tissues from 2 patients, one with preoperative atrial fibrillation and the one with no preoperative atrial fibrillation, were included in the study. The nucleus protein was extracted from the human atrial tissue, and the protein concentration was then measured. A solution with a complex formed through combining magnetic beads with concatenated tandem array of the consensus transcription factor response element DNA sequence (beads-catTFRE) was prepared, and the beads-catTFREs were then used to enrich transcription factors in the nucleoprotein extraction. SDS-PAGE electrophoresis was performed after dissociating beads-catTFRE from nucleoprotein with high temperature and high salt. The gel was then cut and faded before enzymolysis by trypsin in the gels was performed. Acetonitrile was used to extract the peptides from the gels, and the peptide solution was then dried. After that, we dissolved the peptides and performed mass spectrum tests, and the data were analyzed and processed with Firmiana one-stop proteomic analysis platform. In this study, 220 and 181 transcription factors were identified in the normal right atrial tissue and the right atrial tissue with atrial fibrillation, respectively. A total of 241 transcription factors were identified in the two groups. Among the 241 transcription factors, 12 were in the top 10% of those transcription factors that were above the median expression level of the normal right atrial tissue, and 12 transcription factors were in the top 10% of those above the median expression level of the right atrial tissue with atrial fibrillation. The high-throughput profiling method established in this study has high coverage, and the data collected can be used to support further validation studies.

Large-Scale Proteomic Analysis of Follicular Lymphoma Reveals Extensive Remodeling of Cell Adhesion Pathway and Identifies Hub Proteins Related to the Lymphomagenesis.

Simple SummaryFollicular lymphoma represents the major subtype of indolent B-cell non-Hodgkin lymphomas, ranging from about 20 to 30% of all B-NHLs cases in western countries. Yet, the global proteome profile of follicular lymphoma remains largely undocumented; thus, we aimed to employ for the first time a comprehensive proteomic analysis to outline its molecular landscape. A total of 15 lymphoma fine-needle aspiration biopsy samples and 14 controls were evaluated by label-free quantitative proteomics. Among the 7673 proteins identified in our dataset, 1186 proteins were differentially expressed between lymphoma and control samples. Importantly, dysregulated proteins were enriched in biological processes such as B-cell receptor signaling pathway, cellular adhesion molecules pathway, or membrane trafficking. Additionally, we identified several novel hub proteins related to lymphomagenesis. To summarize, we have determined the molecular characteristics of follicular lymphoma and discovered proteins which may hold potential for biomarkers or therapeutic targets.Follicular lymphoma (FL) represents the major subtype of indolent B-cell non-Hodgkin lymphomas (B-NHLs) and results from the malignant transformation of mature B-cells in lymphoid organs. Although gene expression and genomic studies have identified multiple disease driving gene aberrations, only a few proteomic studies focused on the protein level. The present work aimed to examine the proteomic profiles of follicular lymphoma vs. normal B-cells obtained by fine-needle aspiration biopsy (FNAB) to gain deep insight into the most perturbed pathway of FL. The cells of interest were purified by magnetic-activated cell sorting (MACS). High-throughput proteomic profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and allowed to identify of 6724 proteins in at least 75% of each group of samples. The ‘Total Protein Approach’ (TPA) was applied to the absolute quantification of proteins in this study. We identified 1186 differentially abundant proteins (DAPs) between FL and control samples, causing an extensive remodeling of several molecular pathways, including the B-cell receptor signaling pathway, cellular adhesion molecules, and PPAR pathway. Additionally, the construction of protein–protein interactions networks (PPINs) and identification of hub proteins allowed us to indicate the key player proteins for FL pathology. Finally, ICAM1, CD9, and CD79B protein expression was validated in an independent cohort by flow cytometry (FCM), and the results were consistent with the mass spectrometry (MS) data.

High-throughput transcriptomic and proteomic profiling of mesenchymal-amoeboid transition in 3D collagen

The plasticity of cancer cell invasion represents substantial hindrance for effective anti-metastatic therapy. To better understand the cancer cells’ plasticity, we performed complex transcriptomic and proteomic profiling of HT1080 fibrosarcoma cells undergoing mesenchymal-amoeboid transition (MAT). As amoeboid migratory phenotype can fully manifest only in 3D conditions, all experiments were performed with 3D collagen-based cultures. Two previously described approaches to induce MAT were used: doxycycline-inducible constitutively active RhoA expression and dasatinib treatment. RNA sequencing was performed with ribo-depleted total RNA. Protein samples were analysed with tandem mass tag (TMT)-based mass spectrometry. The data provide unprecedented insight into transcriptome and proteome changes accompanying MAT in true 3D conditions.

Application of Proteomics Profiling for Biomarker Discovery in Hypertrophic Cardiomyopathy.

High-throughput proteomics profiling has never been applied to discover biomarkers in patients with hypertrophic cardiomyopathy (HCM). The objective was to identify plasma protein biomarkers that can distinguish HCM from controls. We performed a case-control study of patients with HCM (n = 15) and controls (n = 22). We carried out plasma proteomics profiling of 1129 proteins using the SOMAscan assay. We used the sparse partial least squares discriminant analysis to identify 50 most discriminant proteins. We also determined the area under the curve (AUC) of the receiver operating characteristic curve using the Monte Carlo cross validation with balanced subsampling. The average AUC was 0.94 (95% confidence interval, 0.82-1.00) and the discriminative accuracy was 89%. In HCM, 13 out of the 50 proteins correlated with troponin I and 12 with New York Heart Association class. Proteomics profiling can be used to elucidate protein biomarkers that distinguish HCM from controls.

Abstract A066: Expanding insights into the colorectal cancer tumor proteome; unbiased protein profiling reveals multiple proteomic-based tumor subtypes

Abstract Introduction: Recent approvals of microsatellite instability (MSI) or mismatch repair (MMR) testing, in addition to PD-L1 expression, expand the tools available to identify tumor characteristics that may help predict the likelihood of patient response to immunotherapy treatment. However, even in MSI positive subgroups, not all subjects achieve a durable response and research continues to identify tumor characteristics that further predict the likelihood of patient response. To support and advance this area of research, new tools are being developed that provide deeper and unbiased views of the tumor proteome. Here, we characterize the protein expression profiles of 95 colorectal cancer tumors (CRC) using SWATH acquisition mass spectrometry (SWATH MS) to further probe tumor phenotypic characteristics. Experimental Methods: FFPE colon tissue samples (10 healthy, 95 cancer) were obtained from commercial biobanks. Proteins were extracted from the tissue, processed to peptides with trypsin, and prepared for LC-MS analysis. Peptides for each sample were injected on a Triart C18 column (YMC) coupled to a NanoLC 425 system (SCIEX) using a 43min gradient at a flow rate of 5µl/min. The eluted peptides were then analyzed with a TripleTOF® 6600 system (SCIEX) operated in SWATH mode. Total run time per sample was 1 hour. Data were analyzed in Spectronaut Pulsar X (Biognosys) with a project specific library. All data were filtered with a 1% FDR on peptide and protein level. Results: Across all samples, &amp;gt;4,500 protein groups were quantified (approximately 3,600 per sample). Data analysis revealed a large number of proteins (~1,000) were differentially expressed in the cancer cohort. Consistent with increased tumor cell proliferation, proteins involved in protein translation were upregulated in the tumor samples. Unsupervised clustering of the data separated the healthy and the cancer cohort. Clustering also revealed three main proteomic subtypes within in the cancer cohort (A, B and C), which were largely distinguished by expression of cell adhesion proteins, including neuronal growth regulator 1 (NEGR1), a potential tumor suppressor. Interestingly, hepatocyte nuclear factor 4-alpha (HNF4A), a transcription factor which is known to be elevated in CRC, was only overexpressed in subtype B. Further analysis of key protein networks related to CRC treatment and immunotherapy development will be presented. Conclusions: High-throughput proteomic profiling of FFPE tissues using SWATH-MS enables the deepest phenotypic characterization of tumor tissue. Ultimately, analyses of this type will enable a functional understanding of interplay between the tumor microenvironment, expression of protein networks and response to immune-directed therapies. Citation Format: Nicholas Dupuis, Jan Muntel, Roland Bruderer, Lukas Reiter. Expanding insights into the colorectal cancer tumor proteome; unbiased protein profiling reveals multiple proteomic-based tumor subtypes [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A066.

Lorlatinib Treatment Elicits Multiple On- and Off-Target Mechanisms of Resistance in ALK-Driven Cancer.

: Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non-small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment. SIGNIFICANCE: High-throughput genomic, transcriptomic, and proteomic profiling reveals various mechanisms by which multiple tumor types acquire resistance to the third-generation ALK inhibitor lorlatinib.

High-throughput proteomic profiling of the fish liver following bacterial infection

BackgroundHigh-throughput proteomics was used to determine the role of the fish liver in defense responses to bacterial infection. This was done using a rainbow trout (Oncorhynchus mykiss) model following infection with Aeromonas salmonicida, the causative agent of furunculosis. The vertebrate liver has multifaceted functions in innate immunity, metabolism, and growth; we hypothesize this tissue serves a dual role in supporting host defense in parallel to metabolic adjustments that promote effective immune function. While past studies have reported mRNA responses to A. salmonicida in salmonids, the impact of bacterial infection on the liver proteome remains uncharacterized in fish.ResultsRainbow trout were injected with A. salmonicida or PBS (control) and liver extracted 48 h later for analysis on a hybrid quadrupole-Orbitrap mass spectrometer. A label-free method was used for protein abundance profiling, which revealed a strong innate immune response along with evidence to support parallel rewiring of metabolic and growth systems. 3076 proteins were initially identified against all proteins (n = 71,293 RefSeq proteins) annotated in a single high-quality rainbow trout reference genome, of which 2433 were maintained for analysis post-quality filtering. Among the 2433 proteins, 109 showed significant differential abundance following A. salmonicida challenge, including many upregulated complement system and acute phase response proteins, in addition to molecules with putative functions that may support metabolic re-adjustments. We also identified novel expansions in the complement system due to gene and whole genome duplication events in salmonid evolutionary history, including eight C3 proteins showing differential changes in abundance.ConclusionsThis study provides the first high-throughput proteomic examination of the fish liver in response to bacterial challenge, revealing novel markers for the host defense response, and evidence of metabolic remodeling in conjunction with activation of innate immunity.

Proteomic comparison of selective breeding and growth hormone transgenesis in fish: Unique pathways to enhanced growth

In fish used for food production and scientific research, fast growth can be achieved via selective breeding or induced instantaneously via growth hormone (GH) transgenesis (GHT). The proteomic basis for these distinct routes towards a similar higher phenotype remains uncharacterized, as are associated implications for health parameters. We addressed this knowledge gap using skeletal muscle proteomics in coho salmon (Oncorhynchus kisutch), hypothesising that i) selective breeding and GHT are underpinned by both parallel and unique changes in growth systems, and ii) rapidly-growing fish strains have lowered scope to allocate resources towards immune function. Quantitative profiling of GHT and growth-selected strains was done in comparison to wild-type after injection with PBS (control) or Poly I:C (to mimic infection). We identified remodelling of the muscle proteome in each growth-enhanced strain that was strikingly non-overlapping. GHT was characterized by focal upregulation of systems driving protein synthesis, while the growth-selected fish presented a larger and more diverse set of changes, consistent with complex alterations to many metabolic and cellular pathways. Poly I:C had little detectable effect on the muscle proteome. This study demonstrates that distinct proteome profiles can explain outwardly similar enhanced growth phenotypes, improving our understanding of growth mechanisms in anthropogenic animal strains. SignificanceThis work provides the first proteomic insights into mechanisms underpinning different anthropogenic routes to rapid growth in salmon. High-throughput proteomic profiling was used to reveal changes supporting enhanced growth, comparing skeletal muscle of growth hormone transgenic (GHT) and selectively-bred salmon strains with their wild-type counterparts. Contrasting past mRNA-level comparisons of the same fish strains, our data reveals a surprisingly substantial proteomic divergence between the GHT and selectively bred strains. The findings demonstrate that many unique molecular mechanisms underlie growth-enhanced phenotypes in different types of fish strain used for food production and scientific research.

MiR-30a Remodels Subcutaneous Adipose Tissue Inflammation to Improve Insulin Sensitivity in Obesity.

Chronic inflammation accompanies obesity and limits subcutaneous white adipose tissue (WAT) expandability, accelerating the development of insulin resistance and type 2 diabetes mellitus. MicroRNAs (miRNAs) influence expression of many metabolic genes in fat cells, but physiological roles in WAT remain poorly characterized. Here, we report that expression of the miRNA miR-30a in subcutaneous WAT corresponds with insulin sensitivity in obese mice and humans. To examine the hypothesis that restoration of miR-30a expression in WAT improves insulin sensitivity, we injected adenovirus (Adv) expressing miR-30a into the subcutaneous fat pad of diabetic mice. Exogenous miR-30a expression in the subcutaneous WAT depot of obese mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. High-throughput proteomic profiling and RNA-Seq suggested that miR-30a targets the transcription factor STAT1 to limit the actions of the proinflammatory cytokine interferon-γ (IFN-γ) that would otherwise restrict WAT expansion and decrease insulin sensitivity. We further demonstrated that miR-30a opposes the actions of IFN-γ, suggesting an important role for miR-30a in defending adipocytes against proinflammatory cytokines that reduce peripheral insulin sensitivity. Together, our data identify a critical molecular signaling axis, elements of which are involved in uncoupling obesity from metabolic dysfunction.

A tandem mass tag (TMT) proteomic analysis during the early phase of experimental pancreatitis reveals new insights in the disease pathogenesis

Changes in the protein expression occurring within the initiation phase of acute pancreatitis (AP) might be vital in the development of this complex disease. However, the exact mechanisms involved in the onset of AP remains elusive and most of our knowledge about the pathobiology of AP comes from animal models. We performed in a rat pancreatitic model a high-throughput shotgun proteomic profiling of the soluble and whole membrane fractions from the pancreas during the early phase of cerulein (Cer)-induced AP. We identified 997 proteins, of which 353 were significantly different (22, 276 or 55 in both, the soluble or the membrane fractions, respectively). Gene Ontology and KEGG PATHWAY analyses revealed that these proteins were implicated in molecular mechanisms relevant to AP pathogenesis, including vesicle-mediated and protein transport, lysosomal and mitochondrial impairment or proteolysis. Numerous metabolic processes were downregulated apparently to reduce energy consumption, and a remarkable increase in inflammatory and stress responses was also highlighted. The proteomic data were verified by immunoblotting of 11 and 7 different soluble or membrane-associated proteins, either novel (VPS29 and MCTS1) or known factors in AP. Also, our first observation of the imbalance of some COP proteins during AP early phase deserves further characterization. Biological significanceAP is one of the most important pathological inflammatory states of the exocrine pancreas but its pathophysiology remains incompletely understood, especially the early acinar events. Proteomic analysis of pancreatic subcellular fractions simplifies protein maps and helps in the identification of new protein alterations and biomarkers characterizing pancreatic tissue damage. Our shotgun approach has not been previously used to profile the early proteomic alterations of the disease, which are considered crucial for its development and for the founding of clinical procedures. Furthermore, our subcellular fractionation protocol allowed us to detect changes in membrane proteins so far overlooked in the proteomic study of AP.Accordingly, using TMT proteomics and bioinformatic tools, we were able to detect significant changes in protein expression related to many pathobiological pathways of acute pancreatitis as from the early phase of the disease. To our knowledge, some of these changes, such as the imbalance of some COP proteins, have never been described in this disease.

Filter-Based Protein Digestion (FPD): A Detergent-Free and Scaffold-Based Strategy for TMT Workflows.

High-throughput proteome profiling requires thorough optimization to achieve comprehensive analysis. We developed a filter aided sample preparation (FASP)-like, detergent-free method, termed Filter-Based Protein Digestion (FPD). We compared FPD to protein extraction methods commonly used in isobaric tag-based proteome profiling, namely trichloroacetic acid (TCA) and chloroform-methanol (C-M) precipitation. We divided a mammalian whole cell lysate from the SH-SY5Y neuroblastoma cell line for parallel protein processing with TCA (n = 3), C-M (n = 2), and FPD using either 10 kDa (n = 3) or 30 kDa (n = 3) molecular weight cutoff membranes. We labeled each sample with tandem mass tag (TMT) reagents to construct a TMT11-plex experiment. In total, 8654 proteins were quantified across all samples. Pairwise comparisons showed very little deviation for individual protein abundance measurements between the two FPD methods, whereas TCA and FPD showed the most difference. Specifically, membrane proteins were more readily quantified when samples were processed using TCA precipitation than other methods tested. However, globally, only 4% of proteins differed greater than 4-fold in the most divergent pair of protein extraction methods (i.e., FPD10 and TCA). We conclude that the detergent-free FPD strategy, particularly using the faster-flowing 30 kDa filter, is a seamless alteration to high-throughput TMT workflows.

High throughput and accurate serum proteome profiling by integrated sample preparation technology and single-run data independent mass spectrometry analysis

Mass spectrometry (MS)-based serum proteome analysis is extremely challenging due to its high complexity and dynamic range of protein abundances. Developing high throughput and accurate serum proteomic profiling approach capable of analyzing large cohorts is urgently needed for biomarker discovery. Herein, we report a streamlined workflow for fast and accurate proteomic profiling from 1μL of blood serum. The workflow combined an integrated technique for highly sensitive and reproducible sample preparation and a new data-independent acquisition (DIA)-based MS method. Comparing with standard data dependent acquisition (DDA) approach, the optimized DIA method doubled the number of detected peptides and proteins with better reproducibility. Without protein immunodepletion and prefractionation, the single-run DIA analysis enables quantitative profiling of over 300 proteins with 50min gradient time. The quantified proteins span more than five orders of magnitude of abundance range and contain over 50 FDA-approved disease markers. The workflow allowed us to analyze 20 serum samples per day, with about 358 protein groups per sample being identified. A proof-of-concept study on renal cell carcinoma (RCC) serum samples confirmed the feasibility of the workflow for large scale serum proteomic profiling and disease-related biomarker discovery. Biological significanceBlood serum or plasma is the predominant specimen for clinical proteomic studies while the analysis is extremely challenging for its high complexity. Many efforts had been made in the past for serum proteomics for maximizing protein identifications, whereas few have been concerned with throughput and reproducibility. Here, we establish a rapid, robust and high reproducible DIA-based workflow for streamlined serum proteomic profiling from 1μL serum. The workflow doesn't need protein depletion and pre-fractionation, while still being able to detect disease-relevant proteins accurately. The workflow is promising in clinical application, because the usage of small sample amounts makes blood testing much less invasive, the fully integrated sample preparation by the SISPROT technology greatly improve sample preparation throughput and reproducibility, and the scan feature of DIA method provides a way to convert nonrenewable clinical specimens into permanent digital proteome maps which could be easily reanalyzed.

Targeting AXL and mTOR Pathway Overcomes Primary and Acquired Resistance to WEE1 Inhibition in Small-Cell Lung Cancer.

Purpose: Drugs targeting DNA repair and cell-cycle checkpoints have emerged as promising therapies for small-cell lung cancer (SCLC). Among these, the WEE1 inhibitor AZD1775 has shown clinical activity in a subset of SCLC patients, but resistance is common. Understanding primary and acquired resistance mechanisms will be critical for developing effective WEE1 inhibitor combinations.Experimental Design: AZD1775 sensitivity in SCLC cell lines was correlated with baseline expression level of 200 total or phosphorylated proteins measured by reverse-phase protein array (RPPA) to identify predictive markers of primary resistance. We further established AZD1775 acquired resistance models to identify mechanism of acquired resistance. Combination regimens were tested to overcome primary and acquired resistance to AZD1775 in in vitro and in vivo SCLC models.Results: High-throughput proteomic profiling demonstrate that SCLC models with primary resistance to AZD1775 express high levels of AXL and phosphorylated S6 and that WEE1/AXL or WEE1/mTOR inhibitor combinations overcome resistance in vitro and in vivo Furthermore, AXL, independently and via mTOR, activates the ERK pathway, leading to recruitment and activation of another G2-checkpoint protein, CHK1. AZD1775 acquired resistance models demonstrated upregulation of AXL, pS6, and MET, and resistance was overcome with the addition of AXL (TP0903), dual-AXL/MET (cabozantinib), or mTOR (RAD001) inhibitors.Conclusions: AXL promotes resistance to WEE1 inhibition via downstream mTOR signaling and resulting activation of a parallel DNA damage repair pathway, CHK1. These findings suggest rational combinations to enhance the clinical efficacy of AZD1775, which is currently in clinical trials for SCLC and other malignancies. Clin Cancer Res; 23(20); 6239-53. ©2017 AACR.

Spatiotemporal Proteomic Profiling of Human Cerebral Development

Mass spectrometry (MS) analysis of human post-mortem central nervous system (CNS) tissue and induced pluripotent stem cell (iPSC)-based directed differentiations offer complementary avenues to define protein signatures of neurodevelopment. Methodological improvements of formalin-fixed, paraffin-embedded (FFPE) protein isolation now enable widespread proteomic analysis of well-annotated archival tissue samples in the context of development and disease. Here, we utilize a shotgun label-free quantification (LFQ) MS method to profile magnetically enriched human cortical neurons and neural progenitor cells (NPCs) derived from iPSCs. We use these signatures to help define spatiotemporal protein dynamics of developing human FFPE cerebral regions. We show that the use of high resolution Q Exactive mass spectrometers now allow simultaneous quantification of >2700 proteins in a single LFQ experiment and provide sufficient coverage to define novel biomarkers and signatures of NPC maintenance and differentiation. Importantly, we show that this abbreviated strategy allows efficient recovery of novel cytoplasmic, membrane-specific and synaptic proteins that are shared between both in vivo and in vitro neuronal differentiation. This study highlights the discovery potential of non-comprehensive high-throughput proteomic profiling of unfractionated clinically well-annotated FFPE human tissue from a diverse array of development and diseased states.

Elucidation of the compatible interaction between banana and Meloidogyne incognita via high-throughput proteome profiling.

With a diverse host range, Meloidogyne incognita (root-knot nematode) is listed as one of the most economically important obligate parasites of agriculture. This nematode species establishes permanent feeding sites in plant root systems soon after infestation. A compatible host-nematode interaction triggers a cascade of morphological and physiological process disruptions of the host, leading to pathogenesis. Such disruption is reflected by altered gene expression in affected cells, detectable using molecular approaches. We employed a high-throughput proteomics approach to elucidate the events involved in a compatible banana- M. incognita interaction. This study serves as the first crucial step in developing natural banana resistance for the purpose of biological-based nematode management programme. We successfully profiled 114 Grand naine root proteins involved in the interaction with M. incognita at the 30th- and 60th- day after inoculation (dai). The abundance of proteins involved in fundamental biological processes, cellular component organisation and stress responses were significantly altered in inoculated root samples. In addition, the abundance of proteins in pathways associated with defence and giant cell maintenance in plants such as phenylpropanoid biosynthesis, glycolysis and citrate cycle were also implicated by the infestation.