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Acute ischemic stroke (AIS), a neurological injury resulting from blood clots, is the second most common cause of death worldwide. Novel therapeutics are urgently needed. Rapid developments in instrumentation and bioinformatics have resulted in increased use of mass spectrometry-based proteomics as an effective tool for the in-depth study of AIS. This review focuses on proteomics investigations of AIS in animal models. We highlight the study findings in system-wide protein abundance changes and molecular mechanisms underlying AIS with or without therapeutic intervention, as well as prestroke prognosis investigations. This review reveals that common molecular pathways related to ischemic injury and spontaneous recovery have been uncovered and part of AIS-changed proteins have been repeatedly identified, indicating the promise of proteomics in generating novel therapeutic targets and biomarker candidates. We also discuss challenges, alleviating strategies, and perspectives of mass spectrometry- based proteomics in AIS research and call for a broad application of systems-level investigations on preclinical AIS molecular mechanism elucidation, target discovery and validation, and therapeutics development. Keywords: acute ischemic stroke, ischemic molecular mechanism, MCAO model, proteomics, stroke biomarker AIS, usually triggered by cardiac arrest) or in part of the brain Introduction (focal AIS). Focal AIS is the typical clinical presentation caused Approximately 87% of all strokes are ischemic strokes. Acute by locally formed blood clots or remotely developed and trav- ischemic stroke (AIS) is the second leading cause of death eled plaques. The onset of AIS is triggered unpredictably and worldwide. Over half of affected patients suffer from persistent multifariously, which can result in death or a series of signifi- long-term disability. Although significant improvements have cant injuries in the brain within hours or days. Although many been made in stroke care and prevention, there is very limited pathophysiological and cellular processes, including edema, progress in therapeutic development and biomarker identifica- brain–blood barrier disruption, neuroinflammation, immune tion. Recombinant tissue plasminogen activator (rtPA) is still responses, cell death, neuronal remodeling, and spontaneous the only FDA-approved drug and mechanical thrombectomy is recovery, have been deciphered, the underlying molecular mech- the major technique for treating AIS. Both are only accessible anisms are largely unknown, which impedes the identification to a small fraction of patients due to the strictly eligible criteria of novel targets and biomarkers and thereafter therapeutic and the narrow therapeutic window of a few hours, albeit the development. In addition, the fundamental understanding of accessibility and treatment effect have improved via advanced the etiology of stroke has primarily focused on a single target,  medical devices. The urgent need for new treatments, partic- which is not sufficient to clarify various stroke-induced inju- ularly for limiting cell death and recovering neuronal function ries. Adjunctive therapies paired with reperfusion approaches post AIS, has kept great research interests in the field. Indeed, (eg, mechanical thrombectomy) and cocktail therapies (eg, cell a PubMed search of “(ischemic OR ischemia) AND stroke” therapy) targeting multiple key pathways to simultaneously in [Title/Abstract] (August 2022) included ~75,000 articles reduce neuronal cell death and restore neuroprotection may be (excluding reviews), indicating a big gap from enormous pre- necessitated, further demanding to uncover AIS mechanism at clinical research efforts to a very low success rate of clinical the molecular level. therapy. Among many factors contributing to this disparity, the To delineate molecular mechanisms and identify novel AIS tar- intrinsic complexity of AIS and the lack of novel therapeutic tar- gets and biomarkers, the systems biology (ie, Omics) approach is gets and biomarkers are the leading causes. AIS is a circulatory the best and the most efficient tool. With the rapid development system-linked disorder that can occur in the whole brain (global of instrumentation and bioinformatics, proteomics is capable of globally identifying and quantifying thousands of proteins in Chemical Biology and Proteomics, Biogen, Cambridge, MA, USA a given state of complex biological systems and has become a * Corresponding author: Ru Wei, Chemical Biology and Proteomics, Biogen, valuable tool to divulge molecular pathways and intracellular Cambridge, MA 02142, USA. E-mail: firstname.lastname@example.org signaling cascades leading to new therapeutic targets and bio- Copyright © 2023 The Chinese Medical Association, Published by Wolters markers. Compared with other omics approaches, proteomics Kluwer Health, Inc. This is an open-access article distributed under the terms of has several advantages: proteins perform main and most cellu- the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 lar functions, only via proteomics, one can at omics-scale study (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially protein posttranslational modifications (PTMs), interactions without permission from the journal. and complexes, the crucial players of molecular mechanisms, Journal of Bio-X Research (2023) 6:15–22 and the majority of drug targets are proteins. Owing to their Received: 17 May 2022; Accepted: 11 October 2022 unbiased nature, in-depth proteome coverage, and suitability http://dx.doi.org/10.1097/JBR.0000000000000134 for nearly all biological matrices, mass spectrometry (MS)-based 15 REVIEW ARTICLE Journal of Bio-X Research discovery (or unbiased) proteomics platforms have been widely characterization of the molecular mechanisms underlying AIS used in the systematic investigation of disease mechanisms, tar- progression and facilitation of biomarker and therapeutic target get identification, drug mode of action (MoA) and biomarker discovery. This section focuses on studies of AIS without thera- discovery studies. Proteomics study of AIS is, however, scarce, peutic intervention. fewer than 150 studies were published in the past 20 years and Many pathological changes occur following AIS including neu-  only a small portion of these have relatively in-depth proteome ronal death, inflammation, and oxidative stress. Proteomics coverage (>5000 proteins). has been used to explore the molecular mechanisms associated Research on human AIS is limited owing to high death rate, with AIS-related damages. A comparison of brain tissues from unexpected onset, and severity, mainly focusing on diagnostic/prog- MCAO and sham rats resulted in identification of 282 differen- [2–12] nostic biomarker discovery. Therapeutic targets are commonly tially expressed proteins enriched in energy metabolism and neu- generated and characterized preclinically using various in vitro rodegenerative disease-related pathways. Sod1 and Syn1, which and in vivo models. Simple systems like cell models are suitable are associated with neurodegeneration and synaptic plasticity, for high-throughput screenings and function interrogation. Only were downregulated in ischemic rats, as determined using pro- animal models are capable of closely mimicking pathophysiologi- teomics and western blotting. This finding indicated that these cal conditions in humans and comprehensively assessing responses proteins might be potential therapeutic targets for stroke-in-   of therapeutic intervention, empowering modern drug discovery. duced brain injury. Another study showed that Eno1, a key Testing animals typically have the same genetic background, “per- protein in the glycolytic pathway, was identified using gel-based  fectly” matched age/sex and good survival rates, enabling the use MS as a potential target to alleviate the ischemic injury. Using  of a small number of biological replicates without confounding. a rat model, Chen et al found that ischemia induced oxida- Practically, reproducible stroke lesion size can be only achievable tive and endoplasmic reticulum stress in brain neuron cells by in animals. Accessing various tissues and biofluids is particularly downregulating Comt and Ctsd and upregulating Calb2. Comt important for simultaneously investigating AIS effects extend- and Ctsd were found to be associated with oxidative stress, ing beyond stroke site(s). Indeed, a variety of AIS in vivo models, inflammatory response, and apoptosis. Calb2 was involved mostly mouse or rat and also cynomolgus or rhesus monkey, have in endoplasmic reticulum stress-induced neuronal apoptosis. been developed using diverse AIS induction methods, such as intra- These findings were further validated in vivo and in vitro using [14–17] luminal suture middle cerebral artery occlusion (MCAO), to immunohistochemistry. Label-free proteomics was used to com- recapitulate transient or permanent artery occlusion with complete, pare brain injuries caused by ischemic and hemorrhagic stroke. partial, or no reperfusion, enabling acute, chronic or longitudinal The results showed that 38 and 86 proteins were differentially injury, or recovery from AIS. Detailed discussions about different expressed in ischemic and hemorrhagic stroke, respectively. stroke animal models and applications are out of scope here and Distinct pathological processes were identified with oxidative [18–24] can be found elsewhere. stress and caspase pathways in AIS and autophagy, necrosis, and Proteomic investigations using animal models become vital to calpain activation associated with hemorrhagic stroke, while map molecular events and build knowledge bases of AIS-induced inflammatory and apoptotic effects were associated with both  pathophysiological processes as well as to decipher the MoA of ther- hemorrhagic and ischemic strokes. A study used isobaric tags apeutic interventions. Recent publications have reviewed proteom- for relative and absolute quantitation (iTRAQ)-based proteom- [25–29] ics approaches and their general applications for studying AIS. ics to show that 61 proteins were dysregulated in the acute and/ Herein, this review summarizes the main findings of proteomics or subacute phase(s) poststroke. These proteins were primar- studies of AIS conducted in animal models and provides a snap- ily associated with energy metabolism, glutamate excitotoxic-  shot of insights into molecular mechanisms and repeatedly observed ity, synaptic plasticity, and inflammation. Data independent target/biomarker candidates, serving as a knowledge resource for acquisition (DIA)-based proteomics along with transcriptomics future AIS-related research, demonstrating the potential of pro- showed that the immune response and inflammation played a  teomic approaches, and thus calling for its broad application. key role in stroke progression in the acute phase (6–24 hours). Proteomics analysis using tandem mass tag (TMT) showed that proteins located on organelle outer membranes were downreg- Database retrieval strategy ulated, and those involved in cytosolic the ribosome and spli- Literature review was performed using the PubMed database. ceosomal complex were upregulated, in mice following stroke The search keywords were “acute ischemic stroke,” “AIS,” and 1 hour of reperfusion. These findings suggested that the “ischemic stroke, proteomics,” “therapeutic target and interven- underlying molecular changes in stroke-related damage were tion,” “stroke biomarker,” “MCAO model,” “animal model,”  associated with the recovery process after stroke. PTM pro- and “ischemic molecular mechanism.” The literature search teomics has been increasingly applied in AIS research to under- was performed up to 2022. The articles included in this review stand the molecular mechanisms. Recently, phosphoproteomics were selected based on their relevance to the topic. The results performed on the hippocampus of MCAO mice showed that were further screened according to the title and abstract. Data dysregulated phosphoproteins were closely correlated with the extraction focused on information about the proteomic studies synapse and neurotransmission. Phosphorylation of Syt1 at of acute ischemic stroke in animal models. Thr112, and its interaction with Anxa6, played a key role in ischemia-induced cerebral injury in MCAO mice. The results  Proteomics studies of acute ischemic stroke in were validated in an oxygen-glucose deprivation cell model. Through lysine-lactylation proteomics analysis on rat cerebral animal models  after ischemia followed by reperfusion, Yao et al identified Global proteomic investigation of AIS progression 1003 lactylation sites on 469 proteins, including 54 upregulated Advances in instrumentation and bioinformatic tools have and 54 downregulated lysine-lactylation sites (vs controls) from made MS-based proteomics a valuable tool to globally quan- 49 and 99 proteins, respectively. The authors further validated 2+ tify AIS-induced changes in protein abundance to allow for the altered lactylation on the important Ca signaling proteins 16 Journal of Bio-X Research REVIEW ARTICLE (Scl25a4, Slc25a5, Vdac1, and Vdac2) and proposed lactyla- months poststroke. Nineteen (A2M, APOA2, APOA4, APOD, tion involvement in the underlying mechanism of cerebral isch- C4BPA, C8A, CFD, CFI, CLEC3B, CSPG4, FBLN1, FETUB, emia–reperfusion injury via mediating mitochondrial apoptosis FGA, GPLD1, HPX, RBP4, TF, and VTN) changed at the same   and neuronal death. Using targeted proteomics, molecular direction as those reported in our mouse stroke study. mechanisms that drive the brain-lung interaction poststroke Breakdown of the brain–blood barrier during ischemia leads were studied in a MCAO mouse model. A 92 multiplex protein to leakage of brain-specific proteins into the general circulation. panel developed by Olink Proteomics® was used to analyze the These brain-derived proteins could be potential biomarkers of content in bronchoalveolar lavage fluid and lung homogenates, disease. Some promising protein biomarker candidates such as Hgf, Tgf-α, and Ccl2 were identified and further validated by MMP9, S100B, and CSTA have been proposed for diagnosis the enzyme-linked immunosorbent assay as dysregulated pro- and prognosis of AIS in humans. Efforts have been made to teins in the lungs after cerebral ischemia, suggesting a potential discover additional molecular mechanism-informed AIS bio-  important role in stroke-induced lung damage. markers using animal models. The expression levels of Rhoa Many patients with AIS develop chronic neurological and and Cdc42 increased gradually in the tissues and serum of rats functional disabilities. Therefore, understanding the dynamic during prolonged ischemia (up to 4 hours), as determined using molecular mechanisms associated with AIS is necessary for the iTRAQ-based proteomics and validated using western blot, development of novel therapies to prevent further neuronal which indicated that these proteins may be biomarkers of AIS in  damage and to promote recovery. Proteomics has been used to the acute phases. Proteomics results from the serum of isch- investigate proteome changes in the weeks following AIS. Label- emic rats showed differential expression of A2m, Itih3, C3, Alb, free proteomic analysis of the cortices of MCAO rats from the Hp, and Ttr, which are known to be associated with ischemia. subacute to the chronic phase (days 1, 7, and 14 poststroke) This finding may facilitate future studies aimed at identifica-   found that the expression of 1305 proteins changed during this tion of clinical biomarkers. Datta et al found upregulated period, and cytoskeleton and synaptic structures, energy metab- brain-specific proteins including Gfap, Uchl1, and S100b in the  olism, and inflammatory response were significantly disrupted in poststroke acute and subacute phase. Li et al reported that the subacute phase. However, in the long-term phase, recovery C3, Apoa4, and S100a9 might be potential biomarkers of isch- of the cytoskeleton was detected, and inflammation pathways emia based on increased mRNA and protein expression levels different from those activated during the subacute phase were during stroke progression. Proteomic, immunoblotting, and  activated. Using a distal hypoxic (DH)-MCAO mouse model immunohistological studies showed that Hsp72 was a specific and TMT-based proteomics, our laboratory quantified over 7600 biomarker in the peri-infarct region of rats with permanent focal proteins and found nearly half of them changed in abundance in cerebral ischemia. As a result, an anti-Hsp72 vectorized stealth  MCAO mice during a 28-day poststroke period. Of these, 309 immunoliposome was developed for theranostics of AIS. The were temporally associated with stroke, and underwent relatively 309 sustainably changed proteins identified in our DH-MCAO  large and sustained increases. These proteins were largely associ- study, including 182 annotated as secreted proteins, could ated with the immune response. In addition, proteins involved in serve as candidate pharmacodynamic and diagnostic biomark- cytoskeleton remodeling and synaptic signaling underwent small ers. Increased abundance of some of the 182 proteins was stroke-induced changes. On day 28 poststroke, most of the pro- observed in humans. For example, in a longitudinal biomarker teins returned to normal levels, indicating spontaneous poststroke and drug target study, increases in CFB, AHSG, FN1, and  recovery. A chronic-phase investigation of AIS in cynomolgus APOA1 were observed in human serum with the progression of  monkeys found 55 dysregulated proteins in cortices with elevated AIS. Integration of transcriptomics and proteomics identified or low infarct volumes on day 28 poststroke. These proteins 76 proteins that were differentially expressed in the ischemic were associated with tissue injury and recovery-related cellular brain. Of these, Gadd45g and Ctnnd2 were identified as prom- processes including inflammation, neurogenesis, and synaptogen- ising blood biomarkers for AIS prognosis and diagnosis after  esis. The dysregulated proteins identified poststroke at multiple validation using Parallel Reaction Monitoring (PRM)-targeted time intervals largely overlapped across several studies despite use proteomics and the Nanostring nCounter assay in a new cohort  of different AIS animal models. For example, 604 dysregulated of MCAO mice. Overall, proteomics has identified a pool of  proteins overlapped between the aforementioned rat MCAO biomarker candidates for various poststroke phases. Many bio-  and our mouse DH-MCAO studies, and over 72% of these marker candidates were reported in multiple studies, including proteins changed in the same direction. The cytoskeleton and A2m, Ahsg, Alb, Apoa1, Apoa4, cdc42, Ctsd, Gfap, Hpx, Mug1, [28,33,34,36,41–45,49–56] synaptic remodeling, the adaptive immune response, and possible Rhoa, S100b, Tf, and Ttr. Considering the com- later-phase recovery were observed in both studies. A study by plex physiological changes induced by ischemia, a panel of pro-  Ren et al showed consistent upregulation of 3 proteins (Cdc42, teins might be the most effective AIS biomarker. Eef1a1, and Alb) and downregulation of 13 proteins (Camk2a, MS-based proteomic analysis has identified many known Cntn1, Glud1, Arpc5l, Map6, Ndrg2, Ptpn11, Prkcb, Pgk1, and novel dysregulated proteins from the acute to the long- Prkcg, Srgap3, Uba1, and Nsf) in rats subjected to stroke. In the term phase in AIS, which has allowed for the characterization  previously described monkey study, of 55 stroke-changed pro- of the molecular mechanisms underlying AIS-induced patholog- teins, 16 (CAMK2A, CRMP1, GDA, GPM6A, SYP, A2M, ALB, ical changes such as excitotoxicity, ionic imbalance, oxidative ANXA5, TUBB2A, TUBB4A, COTL1, FLNA, MYH9, TAGLN2, stress, apoptosis, and inflammation. Proteomics results have LDHB, YWHAZ) from the elevated infarct volume group and also provided valuable insights into biomarker and drug target 14 (FABP7, GSN, PFN1, RAB3A, CNN3, S100A11, ATP1A1, identification. TUBA4A, RNH1, PLEC, CALM1, CKB, MDH2, PCP4) from the low infarct volume group changed in the same direction as Proteomic analysis of AIS with therapeutic interventions those in our DH-MCAO mouse study. Furthermore, a human  In addition to research efforts on drug discovery, studies have proteomic study of patients who survived AIS reported changes also focused on other avenues such as dietary supplements, in the expression of 43 proteins in plasma collected at 3 and 12 17 REVIEW ARTICLE Journal of Bio-X Research metabolites, and cell therapy to determine potential therapies Huanwu Decoction treatment reversed levels of most inflamma- for stroke. Several approaches have been shown to mitigate tion and neurodegeneration-associated proteins including A2m, ischemia-induced brain injury. Modulating energy metabolism Serpinb5, Ces1c, Hspa1a, Jup, and Nptxr in the cerebrospi-   and ameliorating oxidative stress have been shown to con- nal fluid. Li et al identified 3216 proteins associated with fer neuroprotection through proteomics analysis. It has been MCAO in mice using iTRAQ-based proteomics, of which 21 found that ferulic acid, a natural antioxidant, exerts a neuro- were differentially expressed following NaoMaiTong treatment. protective effect via attenuation of ischemia-induced changes Enrichment analysis indicated that NaoMaiTong treatment in expression of multiple proteins such as Ahcy, Idh3a, Gapdh, might exert its neuroprotective effects through modulation of [57–60] Ptpa, Hpcal1, Prdx2, and Prdx1. This research group also three pathways: ribosome function (Rpl26, Rpl17, Rpl39, and showed that resveratrol, another antioxidative and anti-inflam- Rps13), tight junctions, and regulation of actin cytoskeleton matory agent, protected against AIS by modulating oxidative (Tuba, Wasl, and Rac1). The expression changes in Rpl17, Tuba,  stress and energy metabolism through increased expression and Rac1 were validated using western blot assay. Proteomics of Prdx5, Idh3a, Apoa1, and Uchl1, and decreased expres- results showed that the therapeutic effects of rhubarb, another  sion Dpysl2. Similarly, retinoic acid was found to perform Chinese traditional medicine, might result from modulation of neuroprotective function by regulating various proteins that various pathways such as cGMP-PKG signaling and the synap- mediate cell metabolism and function such as Ahcy, Idh3a, and tic vesicle cycle via restoration of the expression levels of mul-  Gpd1. A label-free proteomic study found that the tyrosine tiple proteins including Mapk1, Syn1, and Calm1 in MCAO  metabolism and dopaminergic synapse signaling pathways were rats. Furthermore, Eftud2, mTOR, Rab11, Ppp2r5e, Hk1, disrupted in MCAO rats, and glutathione treatment restored and Eno2, which are associated with innate immune regulation, protein expression to normal levels. Western blot validation and mTOR signaling, membrane trafficking, cell growth, and HIF-1 metabolite quantification showed that the mechanism underly- signaling pathways, were identified as key hub proteins underly- ing the therapeutic effects of glutathione on AIS was to increase ing the effects of Hydroxysafflor Yellow A against ischemia-in-  intrastriatal dopamine through reversal of AIS-induced down- duced injury in rats using proteomic analysis. A proteomics regulation of tyrosine hydroxylase. Increased dopamine would study found the tetrandrine isolated from a Chinese analgesic in turn result in increased glutathione through upregulation of medicine alleviated neurological deficits, brain water content,  glutathione synthetase and homocysteine levels. Proteomic and infarct volume by normalizing the expression of Grp78,  analyses using matrix-assisted laser desorption/ionization-time DJ-1, and Hyou1 in MCAO mice. Huanglian Jiedu Decoction of flight (MALDI-TOF) showed that Crmp2, Hsp60, Eno2, Trx, exerts neuroprotective effects via mediating the expressions of and Pp2a, which are involved in energy metabolism, homeo- Grin1, Rap1a, Actb and Akt in the RAP1 signaling pathway, stasis, axonal growth, and oxidative stress, were dysregulated while the beneficial effects of QishenYiqi for stroke recovery in MCAO rats. The levels of these proteins were restored to are closely associated with regulating lysosome pathway and [66,67] normal following treatment with melatonin, a sleep hormone galectin-3-mediated inflammation. Muscone, an active and antioxidant. Further analysis via WB, immunofluorescence, component of musk, ameliorates the neurological damage  and molecular docking confirmed that melatonin attenuated mainly through remedying neuronal synaptic connections. AIS-induced brain damage via regulation multiple protein tar- DIA proteomics results indicated that Alb, mTOR, Stxbp1, Cltc,  gets listed above. Recent proteomics results suggested that Dync1h1, and Sptan1 may be the potential targets of sodium Bexarotene, a FDA-approved drug for cutaneous lymphoma, tanshinone IIA sulfonate, a key ingredient from Salvia miltior-  also displays neuroprotective benefits and inhibits the JIP3/ rhiza Bunge for treating stroke injuries. Scutellarin allevi- ASK1/JNK/Caspase 3 signaling pathway via downregulating ates stroke-induced oxidative stress injury via downregulating JNK-activated scaffolding protein JIP3 to decrease neuronal aldose reductase and its downstream targets Nox1, Nox2, and  apoptosis. Nox4 that lead to reactive oxygen species-induced oxidative  Chinese traditional medicines have been frequently used damage. to alleviate brain injury. MS-based proteomics is an effective Preventive neuroprotection against cerebral ischemia has approach to investigate the MoA of traditional Chinese medi- been studied using MS-based proteomics. The results indicated cines because they have complex compositions. Proteomics anal- that intermittent hypobaric hypoxia preconditioning (6 hours/ ysis using iTRAQ showed that Buyang Huanwu Decoction and day) may have conferred protection in permanent MCAO rats rtPA treatment altered 15 and 23 proteins in mice, respectively. via activation of the clathrin-dependent endocytosis pathway Functional analysis showed that Buyang Huanwu Decoction (upregulation of Chmp1a, Rabep1, Arpc5, and Hspa2) to pro-  treatment led to prevention of brain–blood barrier breakdown mote transport of neuroprotective factors into cells. Using as indicated by restoration of Alb, Fga, and Trf levels, minimi- TMT-based proteomics, 16 proteins associated with neuropro- zation of excitotoxicity through modulation of Gnai1, Gnai2, tective effects were found to be upregulated in MCAO Rhesus Gdi1, and Gdi2, which are associated with GABA receptor monkeys preconditioned with the immune activator D192935 (a activation, and enhancement of energy metabolism through TLR9 agonist). Specifically, F13A1, ORM1/AGP1, and STAB1 inactivation of Gsk-3 and reduced tau activity. In contrast, are associated with M2 macrophages, and MMRN2, STAB1, rtPA induced brain–blood barrier breakdown, as evidenced by HS6ST1, GALNT3, LOXL1 are involved in angiogenesis and  upregulation of Alb, Fga, and Trf, which is a major side effect of tissue repair. Pretreatment with an agonist of PPARα, which  rtPA treatment. To further explore the neuroprotective mech- plays a key role in modulating energy metabolism and vascular anisms of Buyang Huanwu Decoction, this research group com- homeostasis, has also been shown to induce a neuroprotective bined proteomics and metabolomics to comprehensively study effect. A gel-based proteomics study identified 26 dysregulated the cerebrospinal fluid from Buyang Huanwu Decoction-treated proteins in MCAO rats, and PPARα agonist pretreatment pre- MCAO mice. The results showed that disruption of brain–blood served the expression of several proteins such as Ywhaz, Dpysl2, barrier integrity, inflammation, and dysregulated energy homeo- and Snca, which are associated with homeostasis, signal trans- stasis contributed to ischemia-induced brain injury, and Buyang duction, and synaptic plasticity, and modulated the expression 18 Journal of Bio-X Research REVIEW ARTICLE  of Pdia3. Pretreatment with red wine polyphenol compounds addition to preclinical studies, proteomics investigations can be (RWPC) might lead to the neuroprotective effect through mul- conducted using available human samples from other studies tiple biological pathways in rats such as energy metabolism, or biobanks to identify prognostic biomarker candidates. Two  proteolytic pathways, mitochondrial function, and apoptosis. human studies are reviewed in this manuscript to motivate bio- Several other types of interventions also confer neuropro- medical researchers to engage in this area.  tection. Exogenous cell therapy has been evaluated in ischemic Bergerat et al reported global proteomic analysis of cerebral animal models using proteomics. Analysis using MALDI-TOF cortical microvessels from stroke-prone and nonstroke-prone identified 14 proteins that were differentially expressed in stroke rats, and identified about 2000 proteins. Metaproteomic analy- rats with cerebral endothelial cell transplantation compared to sis resulted in identification of differentially regulated proteins the sham and ischemia groups. Further analysis indicated that associated with ischemia, brain–blood barrier integrity, and the neuroprotective effects of this procedure may have resulted angiogenesis and these molecular changes might be related to from controlling neuroinflammation via decreased Spg7 and stroke susceptibility in the prestroke stage. Age and sex were Prdx6, inhibition of a transcriptional repressor via downregu- associated with pathway differences in glycolysis, cell–microen-  lation of Zfp90, and modulation of damaged vasculature via vironment interactions, and transendothelial migration. In a  upregulation of Clic4. Mesenchymal stem cells (MSCs)- spontaneously hypertensive stroke-prone rats (SHRSP) model, derived microvesicles (MVs) have shown the ability to amelio- proteins including Tf, Hpx, Alb, Ashg, Abp, Serpina1, Gc, Ttr, rate functional deficits in permanent MCAO rats. Proteomics and thiostatin were found in the urine and serum weeks before   results showed that treatment with MSC-MVs might have stroke onset. Mitaki et al isolated extracellular vesicles in exerted therapeutic effects on AIS rats through regulation of serum from patients that later developed symptomatic ischemic tissue repair pathways including angiogenesis, neurogenesis, stroke in health checkups and compared the proteomes from  anti-inflammation, and apoptosis. Comprehensive proteomic these vessels against those from sex-matched healthy controls profiling of conditioned media of hiPSC-derived glial and neu- using iTRAQ-based proteomics. The results showed increased ronal progenitor cells (GPCs and NPCs) showed unique pro- expression of several proteins including A2MG, C1QB, C1R, tein expression patterns in each medium, which might explain and HRG, which are involved in the inflammatory and immune  their differential therapeutic effects on AIS rats. Mild hypo- response. These proteins may be potential biomarkers for pre-  thermia is another promising therapeutic strategy for mitigat- diction of future ischemic events. Proteomic analysis of serum ing ischemic injury. A proteomics study found that 26 proteins from patients with AIS and healthy controls using DIA resulted were differentially expressed in ischemic rats with and without in identification of 11 potential protein biomarkers, including hypothermia treatment. These proteins were involved in cellu- F2, VTN, and HRG, that were correlated with one or more lar assembly and organization (Dpysl2, Tuba1a, and Actb), sig- stroke risk factors such as hypertension, cardiovascular disease,  nal transduction (Map2k1, Phb, and Gnao1), and metabolism high cholesterol, and diabetes. (Ndufv2, Bpnt1, and Mdh1), which indicated that hypother- Multiple proteins have been identified as promising indicators mia exerted neuroprotective effects via regulation of multiple of stroke risk, but proteomics research on prestroke prognosis molecular targets and cellular pathways. The authors proposed is underrepresented. This is likely due to the difficulty in predic- Baiap2l1 and A1at as novel therapeutic targets based on their tion of stroke onset and a lack of appropriate animal models. pivotal roles in actin cytoskeleton remodeling and protease inhi- Establishment of biobanks and increased access to human sam-  bition. In another study, label-free proteomics identified 28 ples suitable for investigation of prestroke prognosis will allow upregulated and 22 downregulated proteins in the plasma of for increased numbers of proteomics studies. AIS rhesus monkeys before and after remote ischemic condition- ing. Pathway analysis showed that remote ischemic conditioning Challenges and strategies attenuated brain injury via modulation of multiple pathways related to regulation of lipid metabolism (APOA2 and APOC2), Pharmaceutical industry and AIS research field face mounting anticoagulation (FGA and SERPINA1), complement activation pressure of developing therapeutics to save lives and prevent  (C3 and C1), and endovascular homeostasis (HSPG2). long-term disability and identifying prognostic and diagnos- MS-based proteomics has enabled comprehensive investiga- tic biomarkers to enable stroke prevention screening. From tions of the molecular mechanisms underlying different types of a drug discovery perspective, the lack of comprehensive and therapeutic interventions for AIS, resulting in increased under- in-depth understanding of molecular mechanisms and novel standing of AIS. Revealing neuroprotective MoA of uncon- targets and biomarkers of AIS is the fundamental and most ventional therapeutics, such as Chinese traditional medicine, challenging scientific task. Shifting therapeutic strategies MSC-MV, iPSC-derived GPC, and NPC, and cell conditioning to target multiple pathophysiological processes to limit cell medium, supports their clinical benefits for reducing ischemic death and damage and to promote neuronal recovery are in and reperfusion damage and recovering from AIS, strengthens progress. While it is promising, developing cell therapy has the therapeutic strategy of targeting multiple targets/pathways, been challenging. In addition to improving sustained efficacy, and warrants more continuous efforts in the development of safety and distribution of transplanted cells, systematic under- cocktail therapies for treating AIS. standing of treatment effects and MoA at the molecular level would be crucial for validation and further development of such therapy. The research in this area would largely benefit Prestroke prognosis with MS-based proteomics from extensive proteomics investigation using animal models. As the number of individuals affected by stroke is expected to Additionally, clinical validation of AIS therapeutics proves to increase, improving the prognosis associated with ischemia is be very difficult, as it requires a very large and diverse popula- critical in stroke prevention. Multiple risk factors including dia- tion and so representative of extremely heterogenous clinical betes, atrial fibrillation, hypertension, and dysfunctional glucose presentations and intervention responses of stroke patients. metabolism have been associated with increased risk of AIS. In Recruitment of study subjects is however a big challenge due 19 REVIEW ARTICLE Journal of Bio-X Research to high death and disability rates and short initial treatment Proteomics is a right approach and an effective path forward time window. To this end, rigorous validation of therapeu- to address many challenges in current preclinical research and tics and MoA preclinically and thorough characterization of to build knowledge bases regarding AIS, thus improving and target and biomarker candidates via advanced technologies accelerating AIS drug discovery on multiple targets and path- like proteomics in various preclinical models, particularly in ways. To facilitate its broad application, a throughput leap in animal models, are essential. proteomics sample analysis, standardization and automation On the other hand, proteomics investigation presents its of sample process workflow, as well as an efficient and scal- own challenges. Proteomics is a sophisticated technology that able data mining pipeline are urgently needed. To achieve the generates large data sets and multidimensional biological infor- full potential of proteomics and thereby efficiently promote AIS mation. To achieve excellence in proteomics studies requires a therapeutic development, advanced proteomics technologies, multidisciplinary team of scientists with expertise in biology, including PTM proteomics (eg, phosphorylation and ubiquiti- proteomics, and bioinformatics to correctly design a statisti- nation), interaction proteomics (eg, IP-LC-MS/MS), and che- cally powered study, meticulously execute the sample analy- moproteomics, can be applied to elucidate molecular signaling sis, comprehensively analyze data, and properly interpret the cascades, validate targets, and evaluate intervention effects. results. Lack of proper follow-up for omics studies is a common With the accretion of proteomics data, artificial intelligence will challenge in drug discovery, mostly due to limited resources and become an invaluable tool to mining data and unveiling vital access to advanced technologies. Many proteomics studies have  biological insights. Together with other omics, proteomics generated lists of differentially expressed proteins as potential will accelerate the development of targets, biomarkers, and ther- target and biomarker candidates, but few of these studies have apeutics, and significantly increase the translational significance been able to be used to determine relevant biological informa- of preclinical findings in stroke research. tion. Broad orthogonal validation of the findings of proteomics studies is crucial. Several technologies can be used to facilitate multitarget validation. Targeted proteomics, like PRM, inher- Acknowledgments ently with the highest sensitivity and quantitative accuracy None. among different data acquisition strategies used in MS-based proteomics, are readily accessible in most proteomics labora- tories and can be used to accurately quantify tens to hundreds Author contributions  of proteins at relatively low cost. SomaScan (SomaLogic), FS, RFG, and RW conceived the content and wrote the manu- [83,84] proximity extension assay (Olink), and conventional script. All authors reviewed and approved the final version of [85,86] antibody-based multiplex protein assays/arrays are excel- the manuscript. lent tools for quantification of tens to thousands of proteins. Additional challenges include intrinsic lower sample analy- sis throughput in unbiased proteomics analysis compared to Financial support transcriptomics technologies (eg, RNAseq), low resolution in This work was supported by Biogen. The funder did not par- identifying and distinguishing proteoforms, and relatively low ticipate in data collection and analysis, article writing or sensitivity in quantifying PTM proteins. Technology advance- submission. ments in these areas are steadily progressing but are yet to be revolutionized. Proteomics scientists thus need to vet analytical options and have clearly defined study aims for a proteomic Data availability statement study. Furthermore, the number of reported AIS proteomics Not applicable. studies is still small and they are likely conducted on different preclinical models (eg, different species and stroke induction methods) with different study designs (eg, sampling scheme) Conflicts of interest and various proteomics platforms. Comparison of identified There are no conflicts of interest. No conflicts of interest exist target and/or biomarker candidates across studies is difficult. between Biogen and publication of this paper. More proteomics investigation of AIS is needed, and it is also of great importance to follow the best laboratory practice in this field ranging from sample preparation, instrumentation to References bioinformatics. To take advantage of available proteomics data,  Liaw N, Liebeskind D. 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