Nascent Proteomes in Peripheral Blood Mononuclear Cells as a Novel Source for Biomarker Discovery in Human Stroke
Background and Purpose—The proteome of newly synthesized proteins (nascent proteome) in peripheral blood mononuclear cells (PBMCs) can be a novel source of stroke biomarkers. Changes in the PBMC nascent proteome after stroke reflect the dynamic response-in-action not detectable in the total proteome (all existing proteins) in blood. Here, we test the application of nascent proteomics as a novel approach for stroke biomarker discovery.
Methods—The PBMC nascent proteome in human blood was determined by metabolic labeling of fresh PBMC cultures with azidohomoalanine (an azide-containing methionine surrogate), followed by mass spectrometry detection and quantification of azidohomoalanine-labeled proteins. The PBMC nascent and total proteomes were compared between patients with stroke and matched controls.
Results—Both PBMC nascent and total proteomes showed differences between stroke patients and controls. Results of hierarchical clustering analysis of proteomic data revealed greater changes in the nascent than in the total PBMC proteomes, supporting the usefulness of the PBMC nascent proteome as a novel source of stroke biomarkers.
Conclusions—Nascent proteomes in PBMC can be a novel source for biomarker discovery in human stroke.
The identification of stroke biomarkers has important diagnostic and treatment implications. Recent studies have demonstrated that gene expression profiles in peripheral blood leukocytes can be a source of stroke biomarkers.1,2 Here, we build on this insight and demonstrate that newly synthesized proteins from peripheral blood leukocytes (the nascent proteome) are a novel and revealing source of potential stroke biomarkers.
The nascent proteome of peripheral blood mononuclear cells (PBMC) defines altered protein biosynthesis in the PBMC’s response to an acute event. Essentially, it provides a snap shot of reactive changes that may not be detectable by characterization of the total proteome (consisting of all existing proteins in PBMC). In parallel with analysis of PBMC total proteomes in patients with stroke and controls, we determined nascent proteomes in the same PBMC preparations using a novel approach of metabolically labeling the PBMC fraction with a chemically tagged amino acid, the incorporation of which into newly synthesized proteins allows subsequent isolation and characterization of PBMC nascent proteomes. We found that, compared with the total proteome, the PBMC nascent proteome shows unique bioinformatic features and greater differences between patients with stroke and controls in a sex-specific pattern. This is the first published study investigating the human PBMC nascent proteome as a novel source of stroke biomarkers.
Study Subjects and Methods
Study protocols were approved by Institutional Review Board of the Morehouse School of Medicine and the Grady Memorial Hospital of Atlanta. This pilot study included 4 male and 3 female black patients, admitted to the Marcus Stroke and Neuroscience Center at Grady Memorial Hospital with a clinical diagnosis of stroke, and 5 male and 3 female age-matched black controls, recruited from the Clinical Research Center at Morehouse School of Medicine. Clinical diagnosis of stroke was later verified by review of history, additional clinical examination, and neuroimaging.
From each study subject, 8-mL whole blood was drawn. The average time between known well and study blood draw was 22.9±4.5 (mean±SE) hours. Within 1 hour after blood draw, the PMBC fraction was isolated from the whole blood (methods described in Materials and Methods in the online-only Data Supplement), followed by a 2-hour incubation with azidohomoalanine (an azide-containing methionine surrogate) to metabolically label newly synthesized proteins. After incubation, proteins were extracted from individual PBMC preparations and pooled according to study groups as follows: female patients, male patients, female controls, and male controls. The azidohomoalanine-labeled PBMC proteins (ie, nascent proteome) were isolated from the total proteome by means of the Click reaction3,4 as described in Materials and Methods in the online-only Data Supplement.
Nascent and total proteome preparations were analyzed by quantitative mass spectrometry (MS) with technical replications.5 Technical details for MS analysis using Waters’ Synapt G2S mass spectrometer are introduced in Materials and Methods in the online-only Data Supplement. Bioinformatic analyses of proteomic data were performed with the assistance of commercial bioinformatics tools, as noted in Materials and Methods in the online-only Data Supplement.
Initial MS results were validated by reanalyzing a subset of samples using a second, independent MS system (described in Materials and Methods in the online-only Data Supplement) or by Western blot analysis of selected proteins.
Greater Differences Between Patients With Stroke and Controls in the PBMC Nascent Proteome Than Those in the Total Proteome
In both male and female subjects, common and unique proteins were identified in both stroke and control groups, with more robust unique protein subsets found in PBMC nascent proteomes than those in total proteomes, as demonstrated by Venn diagrams (Figure [A] and [C]). The results of hierarchical clustering analysis of all identified PBMC proteins (Table I in the online-only Data Supplement) revealed the greatest difference in proteomes occurring between the nascent proteomes of male and female stroke groups and the smallest difference between the total proteomes of male and female control groups (Figure [B] and [D]). This demonstrates more profound, sex-specific changes in PBMC nascent proteomes after stroke than in total proteomes. For example, in the PBMC nascent but not in the total proteome, increases were seen for Ras-related protein Rab-10 and integrin linked protein kinase (male patients only), and α enolase, endoplasmin, and protein S100 A9 (female patients only; Table II in the online-only Data Supplement).
Distinct Bioinformatic Properties of the PBMC Nascent Proteome
The PBMC nascent proteome, not the total proteome, was enriched with ribosomal and nuclear proteins (Figure I and Table III in the online-only Data Supplement), whose molecular functions (in gene ontology terms) are of DNA binding, RNA binding, translation regulator activity, or structural molecular activity. A number of these proteins showed an increase in patients with stroke (Table II in the online-only Data Supplement).
In the present study, more substantial changes in PBMC proteomes were detected in patients with stroke and the nascent proteomics approach than those with analysis of the total proteome (Figure). Of interest are the aforementioned proteins that were increased or only detected in the PBMC nascent but not in total proteomes, yielding potential candidates for stroke biomarkers. The observed sex specificity and sex-associated differences in PBMC proteomes in stroke are consistent with prevalence and outcome data, as sexual dimorphism in genomic responses to stroke has been reported.6 Here, the nascent proteomics offers additional tools for investigating underlying mechanisms. Myeloperoxidase has been used as a marker for inflammation and neutrophil infiltration in stroke. A significant association of single-nucleotide polymorphism of myeloperoxidase with stroke risk has been reported in humans.7 Interestingly, in the present study, its increase was detected in both PBMC nascent and total proteomes and in both sexes of patients with stroke (Figures II and III in the online-only Data Supplement; Table II in the online-only Data Supplement). Such results present another potential application of the PBMC nascent proteomics—as a means to understand the response-in-action of reactive protein synthesis in acute brain injury and distinguish changes resulting from increased protein synthesis or degradation. Last but not the least, proteins that were enriched in the PBMC nascent but not in the total proteome, notably those involved in translational machinery (Figure I and Table III in the online-only Data Supplement) and those showing an increase in patients with stroke (Table II in the online-only Data Supplement), offer novel leads for mechanistic studies on the PBMC’s response to stroke.
With limited numbers of study subjects, we are cautious in making definite conclusions on specific proteins as putative stroke biomarkers. However, our quantitative MS analyses are of high reproducibility and reliability, with results confirmed by independent analyses using a second MS system at another facility and by Western blotting (Figures II and III in the online-only Data Supplement).
Our results demonstrate the effectiveness and usefulness of using the nascent proteomics approach to identify PBMC proteins whose changes in stroke have not been reported before or would otherwise not be detected if only the total proteome was analyzed. The methodology established here is being applied in our ongoing studies with expanded numbers of study subjects and inclusion of different stroke subtypes and outcomes.
We thank Eddie Stanley, Xiaoming Chen, and John Klimek for assisting blood specimen collection and protein analyses.
Sources of Funding
This study was supported by grants from National Institute of Health: U54 NS060659 (National Institute for Neurological Disorders and Stroke, Neuroscience Institute, MSM), 1R21NS075538 (Dr Zhou), C06 RR-07571 (National Center for Research Resources, MSM), R25NS065739 (Dr Hall); National Institute of Minority Health and Health Disparities: G12 RR003034, 8U54MD007588-04) (Morehouse School of Medicine); Morehouse School of Medicine Endowment (Dr Simon).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.004576/-/DC1.
- Received December 23, 2013.
- Revision received December 23, 2013.
- Accepted January 6, 2014.
- © 2014 American Heart Association, Inc.
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