Proteomics Of Carotid Atherosclerotic Plaques Based On Data-Independent Acquisition Mass Spectrometry

BackgroundsAtherosclerosis(AS)is a slowly progressive chronic disease,and when an AS plaque ruptures it can lead to a series of acute clinical events,such as myocardial infarction or stroke,which are the leading causes of morbidity and mortality in patients.Among these,unstable carotid plaques increase the risk of ischemic stroke.In recent years,the concept of plaque vulnerability has evolved and some progress has been made in predicting the risk of acute cerebrovascular events.However,both noninvasive imaging techniques and circulating biomarkers currently lack sufficient sensitivity and specificity to be further validated in clinical practice.An in-depth understanding of the molecular mechanisms that contribute to plaque instability and the mechanisms of plaque progression could help identify biomarkers that represent unstable plaques.ObjectiveIn this study,we intend to perform quantitative proteomic analysis based on dataindependent acquisition(DIA)-mass spectrometry(MS)using carotid plaques as the study object,to screen out proteins with significant differences in different types of plaques,and to explore the protein characteristics of different types of plaques,with the aim of finding new player proteins that affect plaque instability and plaque progression.MethodsCarotid plaque specimens and clinical data were first collected from 100 patients who underwent carotid endarterectomy at the Department of Vascular Surgery of Peking Union Medical College Hospital to establish a clinical study cohort.HE staining was performed on the plaque specimens,and the plaques of patients were classified into stable and unstable plaques,AHA Ⅳ,Ⅴ and Ⅵ according to the criteria.In the screening phase,carotid plaques were examined by DIA-MS highthroughput proteomics assay,and 37 stable and 45 unstable plaques,29 AHA Ⅳplaques,20 AHA Ⅴ plaques,and 38 AHA Ⅵ plaques were finally included.Proteins with fold change FC≥2 and P-Value<0.05 were considered as differentially expressed proteins(DEPs).DEPs between different subgroups were screened and bioinformatics analysis of DEPs was performed to explore the molecular pathway mechanisms.In the validation phase,additional carotid plaque specimens from 40 patients were collected to validate the screened differential proteins,again firstly by HE staining,and a total of 20 stable plaques,15 unstable plaques,10 AHA Ⅳplaques,10 AHA Ⅴ plaques,and 15 AHA Ⅵ plaques were included,and positive staining in carotid plaque cross-sections was performed using immunohistochemistry(IHC)The density of cells was quantified to validate the proteomics results.ResultsIn the protein screening phase,of which 3999 proteins were finally included in the screening based on DIA data in the stable group plaques and 397 DEPs in total in the unstable group plaques.Annotation by disease and functional analysis showed that the functions of differential proteins in the stable and unstable groups were mainly focused on cell death and lipid metabolism,and 7 ferroptosis-related proteins(SLC1A5,AIFM2,BID,DPP4,TFR1,TF and GCLC)and 2 lipid metabolism-related proteins(APOA5,CETP)were found to be significantly up-regulated in the unstable plaque group.Inflammation-related protein(CD44)expression was lower in the stable plaque group and significantly elevated in the unstable plaque group.Autophagyassociated protein(MTOR)showed the opposite trend.There was a significant positive correlation between protein CETP and clinical indicator CRP,and between protein GCLC and AIFM2.453 DEPs were screened for AHA Ⅳ and Ⅴ,302 DEPs for AHA Ⅴ and Ⅵ,and 438 DEPs for AHA Ⅳ and Ⅵ.Disease between AHA Ⅳ,Ⅴand Ⅵ plaque proteomes and functional analysis annotations mostly focused on proteins related to apoptosis,autophagy,and cytoskeleton formation,showing different expression intensities,while proteins related to lipid metabolism,cell morphology,protein synthesis,cell movement and function,and cell-to-cell signaling and interactions were activated in each of the three different AHA groups,and a total of 4 proteins showing progressive upregulation in AHA Ⅳ,Ⅴ,and Ⅵ were screened The main functional areas involved include apoptosis,cytoskeleton formation,autophagy,molecular transport,cytoplasmic development,cell spreading,angiogenesis and free radical scavenging.By further evaluation of DEPs,significant differences were found between AHA Ⅳ plaques and AHA Ⅵ plaques for iron homeostasis and cell proliferation migration related proteins(TF,MTOR,SMAD3,TFR1),and immune and inflammatory response related proteins(CD44,DPP4,TREM2,IL-6R,GAL-1).In the protein validation phase,seven ferroptosis-related proteins(SLC1A5,AIFM2,BID,DPP4,TFR1,TF and GCLC)and two lipid metabolism-related proteins(APOA5,CETP)were selected for IHC analysis in the stable and unstable plaque groups.TFR1,TF,AIFM2,DPP4 and GCLC proteins in the unstable plaque The expression of TFR1,TF,AIFM2,DPP4 and GCLC proteins was significantly higher in the fibrous cap region of unstable plaques than in the stable group,while the expression of SLC1A5,BID,CETP and APOA5 proteins was significantly higher in the lipid core region of unstable plaques than in stable plaques,and CETP was not statistically significant.immune and inflammation-related proteins(CD44,TREM2,IL-6R and GAL-1)and cell proliferation.The expression of CD44 and GAL-1 was concentrated in the fibrous cap region,while the expression of TREM2,SMAD3 and IL-6R was significantly decreased.were mainly expressed in the lipid core region.ConclusionIn this study,we used DIA-MS quantitative proteomics analysis to screen proteins,which were experimentally validated by independent sample IHC technique,and found that the expression of proteins SLC1A5,AIFM2,BID,DPP4,TFR1,TF,GCLC,CETP and APOA5 were significantly different between stable and unstable carotid plaques,with CETP and clinical index was a significant correlation between CRP.CD44,TREM2,IL-6R,GAL-1 and SMAD3 proteins were significantly different in AHA Ⅳ and AHA Ⅵ plaques.Thus,we found that ferroptosis,lipid metabolism,immune inflammation and cell proliferation-related factors may play important roles in influencing plaque stability and plaque progression,which provides important ideas for further understanding of the molecular mechanism studies related to plaque progression.