Establishing Quality Control Metrics for Large-Scale Plasma Proteomic Sample Preparation.

Large-scale plasma proteomics studies have been transformed due to the multiplexing and automation of sample preparation workflows. However, these workflows can suffer from reproducibility issues, a lack of standardized quality control (QC) metrics, and the assessment of variation before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The incorporation of robust QC metrics in sample preparation workflows ensures better reproducibility, lower assay variation, and better-informed decisions for troubleshooting. Our laboratory conducted a plasma proteomics study of a cohort of patient samples (N = 808) using tandem mass tag (TMT) 16-plex batches (N = 58). The proteomic workflow consisted of protein depletion, protein digestion, TMT labeling, and fractionation. Five QC sample types (QCstd, QCdig, QCpool, QCTMT, and QCBSA) were created to measure the performance of sample preparation prior to the final LC-MS/MS analysis. We measured <10% CV for individual sample preparation steps in the proteomic workflow based on data from various QC sample steps. The establishment of robust measures for QC of sample preparation steps allowed for greater confidence in prepared samples for subsequent LC-MS/MS analysis. This study also provides recommendations for standardized QC metrics that can assist with future large-scale cohort sample preparation workflows.

Whole blood transcript and protein abundance of the vascular endothelial growth factor family relate to cognitive performance.

The vascular endothelial growth factor (VEGF) family of genes has been implicated in the clinical development of Alzheimer's Disease (AD). A previous study identified associations between gene expression of VEGF family members in the prefrontal cortex and cognitive performance and AD pathology. This study explored if those associations were also observed in the blood. Consistent with previous observations in brain tissue, higher blood gene expression of placental growth factor (PGF) was associated with a faster rate of memory decline (p=0.04). Higher protein abundance of FMS-related receptor tyrosine kinase 4 (FLT4) in blood was associated with biomarker levels indicative of lower amyloid and tau pathology, opposite the direction observed in brain. Also, higher gene expression of VEGFB in blood was associated with better baseline memory (p=0.008). Notably, we observed that higher gene expression of VEGFB in blood was associated with lower expression of VEGFB in the brain (r=-0.19, p=0.02). Together, these results suggest that the VEGFB, FLT4, and PGF alterations in the AD brain may be detectable in the blood compartment.

Establishing Quality Control Procedures for Large-Scale Plasma Proteomics Analyses.

Proteomics research has been transformed due to high-throughput liquid chromatography (LC-MS/MS) tandem mass spectrometry instruments combined with highly sophisticated automated sample preparation and multiplexing workflows. However, scaling proteomics experiments to large sample cohorts (hundreds to thousands) requires thoughtful quality control (QC) protocols. Robust QC protocols can help with reproducibility, quantitative accuracy, and provide opportunities for more decisive troubleshooting. Our laboratory conducted a plasma proteomics study of a cohort of N = 335 patient samples using tandem mass tag (TMTpro) 16-plex batches. Over the course of a 10-month data acquisition period for this cohort we collected 271 pooled QC LC-MS/MS result files obtained from MS/MS analysis of a patient-derived pooled plasma sample, representative of the entire cohort population. This sample was tagged with TMTzero or TMTpro reagents and used to inform the daily performance of the LC-MS/MS instruments and to allow within and across sample batch normalization. Analytical variability of a number of instrumental and data analysis metrics including protein and peptide identifications, peptide spectral matches (PSMs), number of obtained MS/MS spectra, average peptide abundance, percent of peptides with a Δ m/z between ±0.003 Da, percent of MS/MS spectra obtained at the maximum injection time, and the retention time of selected tracking peptides were evaluated to help inform the design of a robust LC-MS/MS QC workflow for use in future cohort studies. This study also led to general tips for using selected metrics to inform real-time troubleshooting of LC-MS/MS performance issues with daily QC checks.

Advancements in automation for plasma proteomics sample preparation.

Automation is necessary to increase sample processing throughput for large-scale clinical analyses. Replacement of manual pipettes with robotic liquid handler systems is especially helpful in processing blood-based samples, such as plasma and serum. These samples are very heterogenous, and protein expression can vary greatly from sample-to-sample, even for healthy controls. Detection of true biological changes requires that variation from sample preparation steps and downstream analytical detection methods, such as mass spectrometry, remains low. In this mini-review, we discuss plasma proteomics protocols and the benefits of automation towards enabling detection of low abundant proteins and providing low sample error and increased sample throughput. This discussion includes considerations for automation of major sample depletion and/or enrichment strategies for plasma toward mass spectrometry detection.

Proteomic changes associated with racial background and sepsis survival outcomes.

Intra-abdominal infection is a common cause of sepsis, and intra-abdominal sepsis leads to ∼156 000 U.S. deaths annually. African American/Black adults have higher incidence and mortality rates from sepsis compared to Non-Hispanic White adults. A limited number of studies have traced survival outcomes to molecular changes; however, these studies primarily only included Non-Hispanic White adults. Our goal is to better understand molecular changes that may contribute to differences in sepsis survival in African American/Black and Non-Hispanic White adults with primary intra-abdominal infection. We employed discovery-based plasma proteomics of patient samples from the Protocolized Care for Early Septic Shock (ProCESS) cohort (N = 107). We identified 49 proteins involved in the acute phase response and complement system whose expression levels are associated with both survival outcome and racial background. Additionally, 82 proteins differentially-expressed in survivors were specific to African American/Black or Non-Hispanic White patients, suggesting molecular-level heterogeneity in sepsis patients in key inflammatory pathways. A smaller, robust set of 19 proteins were in common in African American/Black and Non-Hispanic White survivors and may represent potential universal molecular changes in sepsis. Overall, this study identifies molecular factors that may contribute to differences in survival outcomes in African American/Black patients that are not fully explained by socioeconomic or other non-biological factors.

Benchmarking the proteomic profile of animal models of mesial temporal epilepsy.

OBJECTIVES: We compared the proteomic signatures of the hippocampal lesion induced in three different animal models of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE+HS): the systemic pilocarpine model (PILO), the intracerebroventricular kainic acid model (KA), and the perforant pathway stimulation model (PPS). METHODS: We used shotgun proteomics to analyze the proteomes and find enriched biological pathways of the dorsal and ventral dentate gyrus (DG) isolated from the hippocampi of the three animal models. We also compared the proteomes obtained in the animal models to that from the DG of patients with pharmacoresistant MTLE+HS. RESULTS: We found that each animal model presents specific profiles of proteomic changes. The PILO model showed responses predominantly related to neuronal excitatory imbalance. The KA model revealed alterations mainly in synaptic activity. The PPS model displayed abnormalities in metabolism and oxidative stress. We also identified common biological pathways enriched in all three models, such as inflammation and immune response, which were also observed in tissue from patients. However, none of the models could recapitulate the profile of molecular changes observed in tissue from patients. SIGNIFICANCE: Our results indicate that each model has its own set of biological responses leading to epilepsy. Thus, it seems that only using a combination of the three models may one replicate more closely the mechanisms underlying MTLE+HS as seen in patients.

Automation of On-Resin Enrichment of S-Nitrosylated Proteins for Oxidized Cysteine-Selective cPILOT.

S-Nitrosylation (SNO) is a cysteine post-translational modification that increases with normal aging and is present in Alzheimer's disease and other aging-related illnesses. Detection of SNO-modified proteins can be challenging; however, we previously developed a robust quantitative proteomics approach termed "Oxidized Cysteine-Selective combined precursor isobaric labeling and isobaric tagging (OxcyscPILOT)" that allows for detection of endogenous SNO-modified proteins. OxcyscPILOT involves enrichment of SNO-modified proteins using a thiol-based resin. This enrichment is performed manually, and wash steps with the resin require numerous stages and buffer reagents. The goal of this study is to transfer the manual protocol to an automated liquid handler system in order to reduce wash steps, increase sample throughput, and minimize experimental error. In order to accomplish this, we evaluated the Biomek i7 liquid handler automated workstation and a Positive Pressure ALP (PPA) apparatus to conduct automated on-resin enrichment. Our findings provide starting pressure conditions for the use of PPA in an automated OxcyscPILOT proteomics workflow that could be transferred to other robotic liquid handling systems.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT).

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.