Standardization Of Clinical Laboratory For Human Serum Cystatin C Based On Targeted Quantitative Proteomics

Currently,the incidence of chronic kidney disease(CKD)is increasing year by year,and it has become a public health problem worldwide.The current treatment of Western medicine is mainly aimed at how to prevent or delay the progression of CKD,control clinical symptoms,and prevent various complications.Clinical studies of renin-angiotensin(RAS)system blockers have shown to delay the progression of advanced renal failure(CKD4)There is a certain effect,but there are also obvious limitations.The treatment of CKD with traditional Chinese medicine is mainly based on syndrome differentiation and treatment,and the combination of strengthening and eliminating evil is currently available.The existing research results and clinical practice of traditional Chinese medicine have shown that traditional Chinese medicine has a good effect in the prevention and treatment of CKD,but has not yet seen a high level the evidence proves that Chinese medicine can delay the progression of CKD4 disease.Laboratory medicine can provide traditional Chinese medicine with acceptable evaluation methods and technical standards for western medicine,and then solve the defects of poor repeatability,strong subjectivity and no quantitative indicators of clinical diagnosis and treatment of traditional Chinese medicine.Bridge to communicate with each other.Serum cystatin C(Cys C)is an ideal and reliable endogenous marker to reflect the changes of early glomerular filtration rate(GFR).At present,there are many manufacturers of reagents for clinical detection of serum Cys C,which mainly using immunoassay,and latex enhanced immunoturbi dime try is chosen as the routine method for the determination of Cys C.There is a systematic error in the preliminary evaluation of the performance of reagents from different manufacturers of serum Cys C latex enhanced immunoturbidimetry,resulting in inferior comparability of test results among different detection systems,which can affect the diagnosis of early nephropathy.It is an important measure to realize the equivalence of detection results between different systems through the standardization of clinical laboratory that make the results traceable to the international unit system of(SI)through higher level of primary reference materials and/or reference measurement procedures.The core of standardization work is quantity traceability,which mainly includes two stages:one is to establish the reference system,and the other is to standardize the conventional system with the established reference system,which is to implement the standardization plan.There is no reference method for Cys C in the world;the existing reference materials are not assigned by the reference method and lack of interoperability.ObjectiveUnder this background,our project is based on the conventional methods and reference methods of isotope dilution mass spectrometry(ID/MS)on the basis of creatinine,urea,steroids,vitamins and other projects.The complete structural analysis of Cys C was detected by targeted proteomics technology,and the characteristic peptides were screened.The proteins containing characteristic peptides were labeled with protein expression isotopes as internal standards,the conditions of trypsin hydrolysis and chromatography-mass spectrometry were comprehensively studied,the legal performance was comprehensively evaluated,and the SI traceability of protein quantification was carried out,the accurate quantification of targeted proteins was realized,and the international candidate reference method of serum Cys C was developed.The candidate reference method was used to study the reference materials with interoperability,and then the conventional detection system was evaluated to standardize the detection of serum Cys C.To better clarify the characteristics and content of serum Cys C and its value in the diagnosis of renal diseases.MethodsFirstly,the characteristic peptide of Cys C was analyzed by bioinformatics,and the peptide produced by trypsin hydrolysis of Cys C was predicted by PeptideMass tool in ExPASy software.Then the physical and chemical properties of the characteristic peptides were searched and analyzed by ProtParam tool,including Mr,average hydrophilic coefficient,isoelectric point,acidity and basicity and stability of the hydrolyzed peptides.The characteristic peptides with good stability and solubility were selected as candidate peptides.Then,based on the target molecular information of the selected candidate peptides,the multi-reaction monitoring principle(MRM)is used to select the data for mass spectrometry data acquisition,and to collect the signals that meet the rules of the target ions to remove the interference of the ion signals that do not conform to the rules.According to the quantitative characteristics of characteristic peptides by LC-MS/MS technology,the specific peptides were screened.Cys C fragments and various forms of Cys C chemically denatured by ion modification were preliminarily identified.The Cys C standard sample was hydrolyzed by trypsin,and the hydrolyzed sample was analyzed by high resolution mass spectrometry(HRMS).The separated protein fragments enter the mass spectrometry at different separation times.After the peptides are bombarded by high-speed electrons in the mass spectrometer,they can form ions and break into charged fragments of different sizes,and the breakpoint is mainly at the peptide bonds.High resolution time-of-flight mass spectrometry can determine the mass-charge ratio of the fragments,deduce the mass,and then obtain the amino acid sequence.The amino acid sequence of the fragment was analyzed to verify whether it contained the screened characteristic peptide.Preparation of Cys C internal standard by SILAC:15N labeled Cys C compound was synthesized by prokaryotic expression in E.coli or eukaryotic expression in Pichia pastoris and fermentation and purification techniques.After adding 15N labeled Cys C to the serum sample,it was pretreated,which mainly included the steps of target protein pre-separation,protein denaturation,reduction and alkylation,protein hydrolysis,desalination and purification.The chromatographic and mass spectrometry conditions of ID-LC-MS/MS detection method were systematically optimized.The pretreatment of serum samples was systematically verified,including the removal of high abundance protein,the comparison of different reductants and alkylation reagents,the selection of trypsin,the optimization of the ratio of enzyme to substrate,the optimization of enzyme digestion time,the selection of solid phase extraction column and the evaluation of extraction efficiency,etc.,to determine the optimal sample pretreatment conditions.The sensitivity,specificity,precision,accuracy,linear range and interference of the newly established ID-LC-MS/MS serum Cys C candidate reference method were evaluated,and whether it met the requirement of minimum uncertainty of the reference method was evaluated.The SI traceability study of protein quantification was carried out to achieve the accurate quantification of targeted protein,and the international candidate reference method of serum Cys C was developed.Based on the reference method,the Cys C reference material of human serum matrix was prepared,and then the routine detection system was evaluated to standardize the detection of serum Cys C.To better clarify the characteristics and content of serum Cys C and its value in the diagnosis of renal diseases.ResultsThe purity of the 15N labeled recombinant Cys C protein was more than 95%.Compared with standard Cys C,the 15N labeled recombinant Cys C protein was confirmed by MALDI-TOF MS,and the results were consistent.Characteristic peptide:ALDFAVGEYNK was selected as the characteristic peptide for quantitative detection of Cys C in serum.Mass spectrometry conditions:ESI positive ion mode and multi-reaction monitoring(MRM)mode were selected to analyze the ion channel mass to charge ratio(m/z)of the characteristic peptides of,Cys C,which were 614.1→709.3(quantification)and 614.1→1042.3(identification).The mass to charge ratio(m/z)of the internal standard A[L(13C6,15N)]DFAVGEYNK ion channel was 617.5→709.3(quantification)and 617.5→1042.3(identification).The mass to charge ratio(m/z)of protein internal standard(15N-Cys C)ion channel mass to charge ratio(m/z)of was 619.3→716.3(quantification)and 619.3→1052.3(identification).Chromatographic conditions:Waters ACQUITY UPLC ?BEH,C18,1.7 μm,100 mm × 2.1 mm)column was selected,the organic phase was acetonitrile+0.1%formic acid,the aqueous phase was 0.1%formic acid aqueous solution,and the gradient elution was carried out at a flow rate of 0.3 mL/min.Sample pretreatment conditions:in order to improve the detection sensitivity of the method,Pierce Protein G Magnetic Beads nano-magnetic beads and rabbit polyantibody Cys C(D6U3E)Rabbit mAb were selected to construct nano-immunomagnetic beads to extract Cys C from serum,and then the sample was pretreated according to the steps of protein reduction(TCEP),alkylation(CAA),ultrafiltration,trypsin hydrolysis(enzyme:substrate(w:w)=1:2,digestion for 16 h),SPE purification and so on.Method performance:the linear range of candidate reference method is 0.2 mg/L,1.0 mg/L and 10.0 mg/L,the intra-and inter-assay precision ranges are 1.84%-3.22%and 2.06%-4.33%,respectively.When the concentration is 0.5 mg/L,1.0 mg/L and 5.0 mg/L,the recovery rate of standard sample is 96.1-103.4%,the LOD and LOQ were 0.1 and 0.2 mg/L,respectively.The result of standard reference material ERM-DA471-IFCC was 5.57±0.09(n=3,bias:1.74%)。Reference material:The preliminary prepared serum reference material has good uniformity,stability and interoperability,and the uncertainty of stability in 12 months stored under frozen conditions is less than 1.69%.The relative uncertainty of assignment is 2.31%.The comparability between each routine detection system and the candidate reference method is inferior,and the highest variation is 40.54%.ConclusionThe use of SILAC protein internal standard plays an important role in improving the precision and accuracy of ID-LC-MS/MS method for the detection of Cys C.The optimization of sample pretreatment conditions effectively improves the detection sensitivity of the method.In addition,the mass spectrometry and chromatographic conditions were fully optimized in this study,which provided the basic conditions for the candidate reference method for the detection of human serum Cys C by ID-LC-MS/MS.The initially established candidate reference method has higher precision,linear range and analytical sensitivity,no carrying pollution and high specificity than similar reference methods.A set of standardization process for reference materials,conventional system evaluation and uncertainty evaluation around candidate reference methods has been basically formed,which provides a reference for the standardization practice of other projects.The application of the reference system composed of reference laboratories,reference methods and reference materials is an effective way to standardize the test results.