ABSTRACT
OBJECTIVES@#To study the changes of protein levels in peripheral blood after it dried.@*METHODS@#The proteins from whole blood and bloodstains were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and normalized by the label-free quantification (LFQ) method. The differential proteins were analyzed by using R 4.2.1 software, limma and edgeR package. The analysis of biological function, signaling pathway and subcellular localization for the differential proteins was then performed.@*RESULTS@#A total of 623 and 596 proteins were detected in whole blood and bloodstains, respectively, of which 31 were statistically significant in the quantitative results, including 10 up-regulated and 21 down-regulated proteins in bloodstains.@*CONCLUSIONS@#The protein abundances in whole blood and bloodstains are highly correlated, and the variation of protein abundances may be related to the changes of endogenous and structural proteins in cells. The application of proteomics technology can assist the screening and identification of protein biomarkers, thereby introducing new biomarkers for forensic research.
Subject(s)
Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Proteomics/methods , Blood Stains , BiomarkersABSTRACT
Objective To screen potential DR-related plasma markers by profiling the plasma proteomics in patients with diabetic retinopathy (DR). Methods A total of 8 patients with moderate or higher non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR) enrolled in our hospital are included in the DR group and 6 patients without DR as NDR group. Plasma samples from these 14 patients were subjected to protein-labeled-free quantification and parallel liquid-phase tandem mass spectrometry (LC-MS/MS) in order to calculate the relative protein abundance of each protein detected in plasma. The results were statistically analyzed to find significant differences between the two groups of proteins. Further, bioinformatics analysis was performed, including gene ontology (GO) and Pathway annotation, GO and Pathway enrichment analysis, and protein interaction network analysis. Results A total of 41 differential proteins were identified with mean and median ratios ≥2.5(up/down). Among them, 26 were up-regulated and 15 were down-regulated. GO analysis showed that the binding protein in the differential protein was dominant, the single-organism process in the biological process accounted for the highest proportion, and the cell and cell part in the cell component accounted for the highest proportion. Pathway enrichment analysis indicated that the most significant up-regulated protein, tropomyosin 4 (TPM4), is associated with regulation of muscle contraction; while the most prominent down-regulated protein, platelet membrane glycoproteins V (GPV), plays an important role in extracellular matrix (ECM) receptor interactions. Protein interaction network analysis showed that there were potential interactions among glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sulfhydryl oxidase 1 (QSOX1), immunoglobulin kappa locus (IGK@), TPM4, apolipoproteins C2 (APOC2), and immunoglobulin heavy variable 4-31(IGHV4-31); another potential interaction between pregnancy zone protein (PZP) and tissue inhibitor of metalloproteinase 2 (TIMP2). Conclusion Profiling the proteomics of DR vs NDR with label-free quantification technology successfully identified differentially expressed proteins in DR and NDR. These differential proteins are potential DR associated plasma markers and maybe a new target for early prevention and treatment of DR.
ABSTRACT
By using the high resolution mass spectrometer TripleTOF 5600 , three kinds of standard proteins including bovine serum albumin ( BSA) , ovalbumin ( OVA) and lysozyme C( LYZC) were analyzed, and the correlationship between the ion intensity of mass spectrometry and the relative content of protein sample was investigated. The protein samples were digested by trypsion and diluted to 1-1024 fmol in 7 μL. The ion counts per second ( cps) were used to stand for the amounts of proteins and peptides. Then the correlation between sum of ion intensity ( cps) of all the peptides, number of peptides detected and the amount of proteins was investigated. By comparing the change of values of the same sample in three parallel experiments, a linear relationship between these indexes and the amount of proteins within 1-1024 fmol was found when the cps was more than 1000. Usually, the maximal ion intensity was no more than 1. 5 times of the minimum value for same peptide in triplicate experiments, which suggested that the 3 times or more change of ion intensity was the minimum threshold to determine the differences of proteins amounts in different samples. This study provides a relative quantitative analysis method using qualitative data of high resolution and high scan speed mass spectrometry, which can quickly and easily provide reference for biological and medical research.
ABSTRACT
Wide spread use of Di-(2-ethylhexyl) phthalate (DEHP) has made it a ubiquitous contaminant in today’s environment, responsible for possible carcinogenic and endocrine disrupting effects. In the present investigation an integrative toxico-proteomic approach was made to study the estrogenic potential of DEHP. In vitro experiments carried out with DEHP (0.1-100 μM) induced proliferations (E-screen assay) in human estrogen receptors-α (ERα) positive MCF-7 and ERα negative MDA-MB-231 breast cancer cells irrespective of their ERα status. Further, DEHP suppressed tamoxifen (a potent anti-breast cancer drug) induced apoptosis in both cell types as shown by flowcytometric cell cycle analysis. Label-free quantitative proteomics analysis of the cell secretome of both the cell lines indicated a wide array of stress related, structural and receptor binding proteins that were affected due to DEHP exposure. The secretome of DEHP treated MCF-7 cells revealed the down regulation of lactotransferrin, an ERα responsive iron transport protein. The results indicated that toxicological effects of DEHP did not follow an ERα signaling pathway. However, the differential effects in MCF-7 and MDA-MB-231 cell lines indicate that ERα might have an indirect modulating effect on DEHP induced toxicity.