Proteomics-Based Approach to Identify Novel Blood Biomarker Candidates for Differentiating Intracerebral Hemorrhage From Ischemic Stroke—A Pilot Study

Abstract

Background: A reliable distinction between ischemic stroke (IS) and intracerebral hemorrhage (ICH) is required for diagnosis-specific treatment and effective secondary prevention in patients with stroke. However, in resource-limited settings brain imaging, which is the current diagnostic gold standard for this purpose, is not always available in time. Hence, an easily accessible and broadly applicable blood biomarker-based diagnostic test differing stroke subtypes would be desirable. Using an explorative proteomics approach, this pilot study aimed to identify novel blood biomarker candidates for distinguishing IS from ICH.Material and Methods: Plasma samples from patients with IS and ICH were drawn during hospitalization and were analyzed by using liquid chromatography/mass spectrometry. Proteins were identified using the human reference proteome database UniProtKB, and label-free quantification (LFQ) data were further analyzed using bioinformatic tools.Results: Plasma specimens of three patients with IS and four patients with ICH with a median National Institute of Health Stroke Scale (NIHSS) of 12 [interquartile range (IQR) 10.5–18.5] as well as serum samples from two healthy volunteers were analyzed. Among 495 identified protein groups, a total of 368 protein groups exhibited enough data points to be entered into quantitative analysis. Of the remaining 22 top-listed proteins, a significant difference between IS and ICH was found for Carboxypeptidase N subunit 2 (CPN2), Coagulation factor XII (FXII), Plasminogen, Mannan-binding lectin serine protease 1, Serum amyloid P-component, Paraoxonase 1, Carbonic anhydrase 1, Fibulin-1, and Granulins.Discussion: In this exploratory proteomics-based pilot study, nine candidate biomarkers for differentiation of IS and ICH were identified. The proteins belong to the immune system, the coagulation cascade, and the apoptosis system, respectively. Further investigations in larger cohorts of patients with stroke using additional biochemical analysis methods, such as ELISA or Western Blotting are now necessary to validate these markers, and to characterize diagnostic accuracy with regard to the development of a point-of-care-system for use in resource-limited areas.

Blood-based protein biomarkers for the diagnosis of acute stroke: A discovery-based SWATH-MS proteomic approach

“…The stroke subjects were recruited within an average of 7 days (1–15 days) of symptom onset. Only two proteins (ITIH3 and LBP) were commonly differentially expressed in our study when compared to Malicek et al ( 9 ). Another proteomic study conducted by Allard et al ( 23 ) in plasma samples utilized the SELDI approach and identified four differentially expressed proteins (Apo C-1, Apo C-III, serum amyloid A, and antithrombin-III fragment) between 21 total stroke (IS = 11, ICH = 10) and 21 healthy controls recruited within 72 h. Of these four proteins, serum amyloid A was also differentially expressed in our study and was significantly downregulated in total stroke (fold change= 0.26) compared to the control group.…”

“…No protein was commonly expressed upon comparing their results with our study. The difference in the protein expression profile between our studies could be attributed to the small sample size and longer blood sample collection time in the Malicek et al study (9). Another recent study by Lee et al (7) on serum samples used a similar approach of discovery-based SWATH-MS proteomics and identified 163 differential proteins with more than 2-fold change in 20 IS patients recruited within 10 days of symptom onset compared to 20 healthy controls.…”

“…The most common significant pathways involved complement and coagulation cascades, immune-related processes, acute phase response, acute inflammatory response, hemostasis, and pathways related to extracellular space and matrix. In the Malicek et al ( 9 ) study, they identified 12 significantly differentially expressed proteins between seven stroke and two control subjects in plasma. The stroke subjects were recruited within an average of 7 days (1–15 days) of symptom onset.…”

“…A few studies in the past have used the proteomic approach to identify diagnostic biomarkers in IS compared to healthy controls, but most of these studies recruited IS patients beyond the 24-h time window. In a study published last year by Malicek et al ( 9 ) on plasma samples, four proteins were significantly differentially expressed between three IS and two controls using a label-free proteomic approach. The IS subjects in this exploratory study were recruited within an average duration of 7 days (1–15 days) from symptom onset.…”

“…Few studies in the past have utilized the high-throughput proteomic approaches for blood biomarker identification in stroke (7)(8)(9)(10)(11). However, these studies were conducted beyond the 24-h time window and failed to identify the expression pattern of proteins in the acute phase of stroke.…”