Mass-spectrometry-based proteomic analysis is a powerful approach for discovering new disease biomarkers. However, certain critical steps of study design such as cohort selection, evaluation of ...statistical power, sample blinding and randomization, and sample/data quality control are often neglected or underappreciated during experimental design and execution. This tutorial discusses important steps for designing and implementing a liquid-chromatography-mass-spectrometry-based biomarker discovery study. We describe the rationale, considerations and possible failures in each step of such studies, including experimental design, sample collection and processing, and data collection. We also provide guidance for major steps of data processing and final statistical analysis for meaningful biological interpretations along with highlights of several successful biomarker studies. The provided guidelines from study design to implementation to data interpretation serve as a reference for improving rigor and reproducibility of biomarker development studies.
A new sheathless transient capillary isotachophoresis (CITP)/capillary zone electrophoresis (CZE)–MS interface, based on a commercially available capillary with an integrated metal-coated ESI ...emitter, was developed in this study aiming at overcoming the reproducibility and ruggedness problems suffered to a certain degree by almost all the available CE–MS interfaces, and pushing the CE–MS technology suitable for routine sample analysis with high sensitivity. The new CITP/CZE–MS interface allows the electric contact between ESI voltage power supply and the CE separation liquid by using a conductive liquid that comes in contact with the metal-coated surface of the ESI emitter, making it a true sheathless CE–MS interface. Stable electrospray was established by avoiding the formation of gas bubbles from electrochemical reaction inside the CE capillary. Crucial operating parameters, such as sample loading volume, flow rate, and separation voltage, were systematically evaluated for their effects on both CITP/CZE separation efficiency and MS detection sensitivity. Around one hundred CITP/CZE–MS analyses can be easily achieved by using the new sheathless CITP/CZE interface without a noticeable loss of metal coating on the ESI emitter surface, or degrading of the ESI emitter performance. The reproducibility in analyte migration time and quantitative performance of the new interface was experimentally evaluated to demonstrate a LOQ below 5 attomole.
Lipidomics is a critical part of metabolomics and aims to study all the lipids within a living system. We present here the development and evaluation of a sensitive capillary UPLC-MS method for ...comprehensive top-down/bottom-up lipid profiling. Three different stationary phases were evaluated in terms of peak capacity, linearity, reproducibility, and limit of quantification (LOQ) using a mixture of lipid standards representative of the lipidome. The relative standard deviations of the retention times and peak abundances of the lipid standards were 0.29% and 7.7%, respectively, when using the optimized method. The linearity was acceptable at >0.99 over 3 orders of magnitude, and the LOQs were sub-fmol. To demonstrate the performance of the method in the analysis of complex samples, we analyzed lipids extracted from a human cell line, rat plasma, and a model human skin tissue, identifying 446, 444, and 370 unique lipids, respectively. Overall, the method provided either higher coverage of the lipidome, greater measurement sensitivity, or both, when compared to other approaches of global, untargeted lipid profiling based on chromatography coupled with MS.
Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development ...of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)–based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano–liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50–100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.
Two-dimensional reversed-phase capillary liquid chromatography (2D RPLC) separations have enabled comprehensive proteome profiling of biological systems. However, milligram sample quantities of ...proteins are typically required due to significant losses during offline fractionation. Such a large sample requirement generally precludes the application samples in the nanogram to low-microgram range. To achieve in-depth proteomic analysis of such small-sized samples, we have developed the nanoFAC (nanoflow Fractionation and Automated Concatenation) 2D RPLC platform, in which the first dimension high-pH fractionation was performed on a 75-μm i.d. capillary column at a 300 nL/min flow rate with automated fraction concatenation, instead of on a typically used 2.1 mm column at a 200 μL/min flow rate with manual concatenation. Each fraction was then fully transferred to the second-dimension low-pH nanoLC separation using an autosampler equipped with a custom-machined syringe. We have found that using a polypropylene 96-well plate as collection device as well as the addition of n-Dodecyl β-d-maltoside (0.01%) in the collection buffer can significantly improve sample recovery. We have demonstrated the nanoFAC 2D RPLC platform can achieve confident identifications of ∼49,000–94,000 unique peptides, corresponding to ∼6,700–8,300 protein groups using only 100–1000 ng of HeLa tryptic digest (equivalent to ∼500–5,000 cells). Furthermore, by integrating with phosphopeptide enrichment, the nanoFAC 2D RPLC platform can identify ∼20,000 phosphopeptides from 100 μg of MCF-7 cell lysate.
Objective
Previous gene expression analysis identified a network of coexpressed genes that is associated with β‐amyloid neuropathology and cognitive decline in older adults. The current work targeted ...influential genes in this network with quantitative proteomics to identify potential novel therapeutic targets.
Methods
Data came from 834 community‐based older persons who were followed annually, died, and underwent brain autopsy. Uniform structured postmortem evaluations assessed the burden of β‐amyloid and other common age‐related neuropathologies. Selected reaction monitoring quantified cortical protein abundance of 12 genes prioritized from a molecular network of aging human brain that is implicated in Alzheimer's dementia. Regression and linear mixed models examined the protein associations with β‐amyloid load and other neuropathological indices as well as cognitive decline over multiple years preceding death.
Results
Average age at death was 88.6 years. Overall, 349 participants (41.9%) had Alzheimer's dementia at death. A higher level of PLXNB1 abundance was associated with more β‐amyloid load (p = 1.0 × 10−7) and higher PHFtau tangle density (p = 2.3 × 10−7), and the association of PLXNB1 with cognitive decline is mediated by these known Alzheimer's disease pathologies. On the other hand, higher IGFBP5, HSPB2, and AK4 and lower ITPK1 levels were associated with faster cognitive decline, and, unlike PLXNB1, these associations were not fully explained by common neuropathological indices, suggesting novel mechanisms leading to cognitive decline.
Interpretation
Using targeted proteomics, this work identified cortical proteins involved in Alzheimer's dementia and begins to dissect two different molecular pathways: one affecting β‐amyloid deposition and another affecting resilience without a known pathological footprint. Ann Neurol 2018;83:78–88
Acute muscle injuries are exceedingly common and non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed to reduce the associated inflammation, swelling and pain that peak 1-2 days ...post-injury. While prophylactic use or early administration of NSAIDs has been shown to delay muscle regeneration and contribute to loss of muscle strength after healing, little is known about the effects of delayed NSAID use. Further, NSAID use following non-penetrating injury has been associated with increased risk and severity of infection, including that due to group A streptococcus, though the mechanisms remain to be elucidated. The present study investigated the effects of delayed NSAID administration on muscle repair and sought mechanisms supporting an injury/NSAID/infection axis.
A murine model of eccentric contraction (EC)-induced injury of the tibialis anterior muscle was used to profile the cellular and molecular changes induced by ketorolac tromethamine administered 47 hr post injury.
NSAID administration inhibited several important muscle regeneration processes and down-regulated multiple cytoprotective proteins known to inhibit the intrinsic pathway of programmed cell death. These activities were associated with increased caspase activity in injured muscles but were independent of any NSAID effect on macrophage influx or phenotype switching.
These findings provide new molecular evidence supporting the notion that NSAIDs have a direct negative influence on muscle repair after acute strain injury in mice and thus add to renewed concern about the safety and benefits of NSAIDS in both children and adults, in those with progressive loss of muscle mass such as the elderly or patients with cancer or AIDS, and those at risk of secondary infection after trauma or surgery.
Penicillin binding protein 2a (PBP2a)-dependent resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is regulated by the activity of the tricarboxylic acid (TCA) ...cycle via a poorly understood mechanism. We report that mutations in sucC and sucD, but not other TCA cycle enzymes, negatively impact β-lactam resistance without changing PBP2a expression. Increased intracellular levels of succinyl coenzyme A (succinyl-CoA) in the sucC mutant significantly perturbed lysine succinylation in the MRSA proteome. Suppressor mutations in sucA or sucB, responsible for succinyl-CoA biosynthesis, reversed sucC mutant phenotypes. The major autolysin (Atl) was the most succinylated protein in the proteome, and increased Atl succinylation in the sucC mutant was associated with loss of autolytic activity. Although PBP2a and PBP2 were also among the most succinylated proteins in the MRSA proteome, peptidoglycan architecture and cross-linking were unchanged in the sucC mutant. These data reveal that perturbation of the MRSA succinylome impacts two interconnected cell wall phenotypes, leading to repression of autolytic activity and increased susceptibility to β-lactam antibiotics. IMPORTANCE mecA-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene, sucC, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome. Although cell wall structure and cross-linking were unchanged, significantly increased succinylation of the major autolysin Atl, which was the most succinylated protein in the proteome, was accompanied by near complete repression of autolytic activity. These findings link central metabolism and levels of succinyl-CoA to the regulation of β-lactam antibiotic resistance in MRSA through succinylome-mediated control of two interlinked cell wall phenotypes. Drug-mediated interference of the SucCD-controlled succinylome may help overcome β-lactam resistance.
Our study details the stepwise evolution of gilteritinib resistance in FLT3-mutated acute myeloid leukemia (AML). Early resistance is mediated by the bone marrow microenvironment, which protects ...residual leukemia cells. Over time, leukemia cells evolve intrinsic mechanisms of resistance, or late resistance. We mechanistically define both early and late resistance by integrating whole-exome sequencing, CRISPR-Cas9, metabolomics, proteomics, and pharmacologic approaches. Early resistant cells undergo metabolic reprogramming, grow more slowly, and are dependent upon Aurora kinase B (AURKB). Late resistant cells are characterized by expansion of pre-existing NRAS mutant subclones and continued metabolic reprogramming. Our model closely mirrors the timing and mutations of AML patients treated with gilteritinib. Pharmacological inhibition of AURKB resensitizes both early resistant cell cultures and primary leukemia cells from gilteritinib-treated AML patients. These findings support a combinatorial strategy to target early resistant AML cells with AURKB inhibitors and gilteritinib before the expansion of pre-existing resistance mutations occurs.
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•Stepwise model of early to late gilteritinib resistance recapitulates human disease•Early resistant cells in marrow microenvironment rely on AURKB to resume growth•Pre-existing NRAS mutations expand in late resistance and drive relapse•Metabolic reprogramming occurs during evolution of gilteritinib resistance
Gilteritinib is an effective FLT3 inhibitor for AML, but residual cells survive in the marrow microenvironment. Over time, these early resistant cells evolve intrinsic mechanisms of resistance leading to relapse. Joshi et al. use a comprehensive approach to interrogate the evolution of resistance; identifying AURKB as critical for early resistance.
The human urinary proteome provides an assessment of kidney injury with specific biomarkers for different kidney injury phenotypes. In an effort to fully map and decipher changes in the urine ...proteome and peptidome after kidney transplantation, renal allograft biopsy matched urine samples were collected from 396 kidney transplant recipients. Centralized and blinded histology data from paired graft biopsies was used to classify urine samples into diagnostic categories of acute rejection, chronic allograft nephropathy, BK virus nephritis, and stable graft. A total of 245 urine samples were analyzed by liquid chromatography–mass spectrometry using isobaric Tags for Relative and Absolute Quantitation (iTRAQ) reagents. From a group of over 900 proteins identified in transplant injury, a set of 131 peptides were assessed by selected reaction monitoring for their significance in accurately segregating organ injury causation and pathology in an independent cohort of 151 urine samples. Ultimately, a minimal set of 35 proteins were identified for their ability to segregate the 3 major transplant injury clinical groups, comprising the final panel of 11 urinary peptides for acute rejection (93% area under the curve AUC), 12 urinary peptides for chronic allograft nephropathy (99% AUC), and 12 urinary peptides for BK virus nephritis (83% AUC). Thus, urinary proteome discovery and targeted validation can identify urine protein panels for rapid and noninvasive differentiation of different causes of kidney transplant injury, without the requirement of an invasive biopsy.
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