| [Objective]1.To explore the proteomic changes during the formation of dried blood spots.2.To discuss the proteomic stability of dried blood spots over different storage times.3.To identify the stability of blood-specific and age-related protein biomarkers in peripheral blood-original samples.[Methods]1.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)and label-free quantitative(LFQ)with high resolution,benefiting performance and good stability were selected according to the experimental purpose.2.After trypsin treatment,the peptides from whole blood,dried blood spots,plasma and blood cells were subjected to mass spectrometry,the raw files were obtained.After matching with the sample-specific database,quality control analysis at the peptide and protein levels was performed,and then,according to the quantitative results,whole blood and blood spots proteomics were compared and analyzed by the R package limma,next,the biological function,signaling pathway and subcellular localization of the differential proteins were analyzed to explore the potential mechanism of protein abundance change during the blood drying.3.The dried blood spots were deposited at room temperature for 0,10,20 days,1,2,3,and 6 months before stored at ultra-low temperature.The differences in protein numbers and abundance in each pair of groups were analyzed after measured by mass spectrometry.4.After collecting 12 blood-specific and 13 age-related biomarkers from literatures,the stability in whole blood and dried blood spots of these biomarkers were assessed.[Results]1.In this study,by mass spectrometry the total number of peptides identified in peripheral blood was 7348,which includes 7006 specific peptides.Under the requirements that the false discovery rate(FDR)was less than 1%,the identified protein with at least one specific peptide supported,and detected in at least two biological replicates,623,596,258,and 485 proteins were identified in whole blood,blood spots,plasma,and blood cells,respectively.2.An overlap of 572 proteins were detected in whole blood and blood spots,and the Pearson correlation coefficient was 0.9926.With a fold change(FC)of at least 1.5 and adjust P values less than 0.05,15 proteins were detected with significant difference between whole blood and blood spots,seven of which were up-regulated in blood spots and eight of which were down-regulated in blood spots.3.The qualitative and quantitative results of blood spot proteomics within 6 months showed stable expression.The Pearson correlation coefficient in each pair of groups was all greater than 0.99.Except for FKBP1A,no significant correlation was detected in other proteins in their abundance with storage time.4.The detection rates of blood-specific and age-related biomarkers are 66.7%and 23.1%,respectively.The protein abundance of the detectable biomarkers is not significant different between whole blood and blood spots.[Conclusions]1.The method of measuring proteomics in peripheral blood-original samples by mass spectrometry has high sensitivity,good qualitative and quantitative reproducibility and high consistency.The proteomics of whole blood and blood spots has a strong correlation,and the proteomics varied greatly between plasma and other samples.2.Most proteins are stable when dried blood spots formed from peripheral blood,abundance changes in a few proteins between whole blood and blood spots correlated with changes in endogenous and structural proteins in cells.3.There is no significant difference in the proteomics of dried blood spots stored for 6 months at room temperature,and their quality could meet the requirements of downstream experiments as well as forensic identification.4.Peripheral blood-specific protein biomarkers had a high detection rate and were stably expressed in whole blood and blood spots,which might be used for peripheral blood identification in forensic science.Age-related biomarkers had a high omission ratio and it could be recommended as a complementary assay in forensic age estimation. |
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