Proteomic changes associated with racial background and sepsis survival outcomes

Kapp, Kathryn L; Arul, Albert B.; Zhang, Kevin C; Du, Liping; Yende, Sachin; Kellum, John A.; Angus, Derek C.; Peck-Palmer, Octavia M; Robinson, Renã A. S.

doi:10.1039/d2mo00171c

Cited by 5 publications

(18 citation statements)

References 114 publications

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“…Briefly, samples were separated with a 96 min gradient profile: 0–7 min, 0% mobile phase B; 7–13 min, 0–16% B; 13–73 min, 16–40% B; 73–77 min, 40–44% B; 77–82 min, 44–60% B; and 82 to 96 min, 60% B, where mobile phase A was 4.5 mM ammonium formate and 2% ACN (pH 10) and mobile phase B was 4.5 mM ammonium formate and 90% ACN (pH 10). Eluent was concatenated into 24 fractions and concentrated using a SpeedVac (Thermo Scientific) . Before and between each batch injection, a blank injection of mobile phase A and a QC injection of a bovine serum albumin digest occurred.…”

Section: Methodsmentioning

confidence: 99%

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Evaluating cPILOT Data toward Quality Control Implementation

Bowser

Patterson

Robinson

2023

J. Am. Soc. Mass Spectrom.

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Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most mass-spectrometry-based proteomics workflows, multiplexing is achieved by labeling biological samples with heavy isotopes via precursor isotopic labeling or isobaric tagging. Enhanced multiplexing strategies, such as combined precursor isotopic labeling and isobaric tagging (cPILOT), combine multiple technologies to afford an even higher sample throughput. Critical to enhanced multiplexing analyses is ensuring that analytical performance is optimal and that missingness of sample channels is minimized. Automation of sample preparation steps and use of quality control (QC) metrics can be incorporated into multiplexing analyses and reduce the likelihood of missing information, thus maximizing the amount of usable quantitative data. Here, we implemented QC metrics previously developed in our laboratory to evaluate a 36-plex cPILOT experiment that encompassed 144 mouse samples of various tissue types, time points, genotypes, and biological replicates. The evaluation focuses on the use of a sample pool generated from all samples in the experiment to monitor the daily instrument performance and to provide a means for data normalization across sample batches. Our results show that tracking QC metrics enabled the quantification of ∼7000 proteins in each sample batch, of which ∼70% had minimal missing values across up to 36 sample channels. Implementation of QC metrics for future cPILOT studies as well as other enhanced multiplexing strategies will help yield high-quality data sets.

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Section: Methodsmentioning

confidence: 99%

“…Eluent was concatenated into 24 fractions and concentrated using a SpeedVac (Thermo Scientific). 44 Before and between each batch injection, a blank injection of mobile phase A and a QC injection of a bovine serum albumin digest occurred.…”

Section: Basic Phmentioning

confidence: 99%

Evaluating cPILOT Data toward Quality Control Implementation

Bowser

Patterson

Robinson

2023

J. Am. Soc. Mass Spectrom.

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“…A total of 166 patients were selected for this study. Plasma samples were collected at "time zero," which referred to the point of ED admission and enrollment in the ProCESS cohort before treatment, and survival outcomes refer to a patient's status 90 days after ED admission (39). Treatment group was not included as a covariate in this study (43).…”

Section: Ethics Statement Study Design Patients and Plasma Collectionmentioning

confidence: 99%

“…Peptide samples (25 μg) were reconstituted in 100 mM triethyl ammonium bicarbonate and labeled with a TMTpro reagent following the manufacturer's protocol in a 1:8 sample:tag ratio (Thermo Fisher Scientific; Waltham, MA). Samples in each batch were pooled, acetonitrile solvent was removed with centrifugal evaporation, and peptides were desalted as previously described (39). Pooled TMTpro batches (295 μg) were fractionated using basic RPLC offline fractionation to a final set of 24 concatenated fractions (39,45).…”

Section: Sample Taggingmentioning

confidence: 99%

“…Samples in each batch were pooled, acetonitrile solvent was removed with centrifugal evaporation, and peptides were desalted as previously described (39). Pooled TMTpro batches (295 μg) were fractionated using basic RPLC offline fractionation to a final set of 24 concatenated fractions (39,45). Each fraction (1 μg) was reconstituted in LC-MS grade water with 0.1% formic acid (FA) and stored at −80°C.…”

Section: Sample Taggingmentioning

confidence: 99%

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Pathways Associated with Positive Sepsis Survival Outcomes in African American/Black and Non-Hispanic White UTI Patients

Kapp

Choi

Bai

et al. 2023

Shock

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Urinary tract infections (UTIs) are a common cause of sepsis worldwide. Annually, over 60,000 US deaths can be attributed to sepsis secondary to UTIs, and African American/Black adults have higher incidence and case-fatality rates more so than Non-Hispanic White adults. Molecular-level factors that may help partially explain differences in sepsis survival outcomes between African American/Black and Non-Hispanic White adults are not clear. In this study, patient samples (N = 166) from the Protocolized Care for Early Septic Shock (ProCESS) cohort were analyzed using discovery-based plasma proteomics. Patients had sepsis secondary to UTIs and were stratified according to self-identified racial background and sepsis survival outcomes. Proteomics results suggest patient heterogeneity across mechanisms driving survival from sepsis secondary to UTIs. Differentially-expressed proteins (N = 122, FDR-adjusted p < 0.05) in Non-Hispanic White sepsis survivors were primarily in immune system pathways, while differentially-expressed proteins (N = 47, FDR-adjusted p < 0.05) in African American/Black patients were mostly in metabolic pathways. However, in all patients, regardless of racial background, there were 16 differentially-expressed proteins in sepsis survivors involved in translation initiation and shutdown pathways. These pathways are potential targets for prognostic intervention. Overall, this study provides information about molecular factors that may help explain disparities in sepsis survival outcomes among African American/Black and Non-Hispanic White patients with primary UTIs.

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

Oliver,

Choi,

Arul

et al. 2024

ACS Meas. Sci. Au

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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.

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Proteomic changes associated with racial background and sepsis survival outcomes

Abstract: Plasma proteomics identified proteins in various immune pathways that may contribute to racial/ethnic disparities in sepsis survival outcomes.

Cited by 5 publications

References 114 publications

Evaluating cPILOT Data toward Quality Control Implementation

Evaluating cPILOT Data toward Quality Control Implementation

Pathways Associated with Positive Sepsis Survival Outcomes in African American/Black and Non-Hispanic White UTI Patients

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

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