Serum Proteomics Screening And Target Protein Biomarkers Verifying On Uyghur MODY Pedigrees

Objective: Maturity onset diabetes of the young(MODY)is a monogenic autosomal dominant form of diabetes mellitus.Due to MODY subtypes are clinically and hereditarily heterogeneous,MODY always been misdiagnosed as type 1 diabetes(T1DM)and type 2 diabetes(T2DM).Mostly gene mutations in MODY could lead to nonsynonymous changes in their protein products,those mutated proteins are deduced to have a mutated residue that possibly disturbs the metabolism homeostasis and brings consequent alternations in the body fluid components of MODY patients.We proposed that the protein mutations of the MODY patients come from the same family could disturb the changes of protein abundance in both tissues and cells,including the MODY-related responses in serum.Herein,we will explore MODY-specific serum target protein by a Uyghur MODY pedigree based on iTRAQ and MRM proteomics methods.Methods: We took the serum samples from a Uyghur MODY family and used a quantitative proteomic approach with isobaric labeling(iTRAQ)to examine changes in the serum proteome of 4 patients with typical clinical symptoms of MODY compared with 4 healthy control.After depleting of abundant proteins were digested with trypsin and labeled with iTRAQ reagents.Then MS analysis was performed with LC–MS/MS serum profiling.The differential expressed proteins were defined after database research.The selected candidates were further verified in the 8 individual serum samples both MODY and control using MRM-based targeting analysis.Still with MRM,the MODY-related indicative potentials were quantitatively evaluated in the individual serum samples of T1 D,T2D and MODY for looking for their specificity to MODY.Results: Mass spectrometry data were searched by Mascot and PD software,and the absolute value of the logarithm of the ratio of protein content was used as the critical point,and the quantitative analysis of MODY was performed in iTRAQ cases and control.In total,293 serum proteins with 2551 unique peptides were identified.Then a total of 32 differential proteins,including 14 up-regulated and 18 down-regulated were discovered.Besides the 32 differential proteins naturally became the MRM targets,another 9 proteins,which are believed diabetic involvement were not identified as the differential proteins in the iTRAQ experiment,were selected for the MRM verification,such as CATG,LG3 BP,POSIN,FIBG,CO6,FA5,MASP2,CD5 L and KV110.A total of 41 candidates were thus chosen as the target proteins for MRM assay.Since all the abundance changes in these candidates were globally identified in all the individual MODY patients,the target proteomics well verified the 12 potential serum biomarkers for MODY that were originally discovered by iTRAQ as mentioned above.With the strict criterion,the 6 proteins were identified as down-regulated,including SERPINA7,CD14,CETP,PZP,SERPIND1,and SHBG,and another 6 were detected as up-regulated in the MODY sera,including APOC4,SERPINA1,CRP,LPA,C6 and F5.A comparison experiment was implemented to evaluate the abundance levels of the 12 serum proteins in the T1 D,T2D,MODY and healthy people.Totally 30 individual serum samples were collected,including 10 T1 D,10 T2 D and 10 healthy people with age and gender-matched to diabetes cases.Our strategy is divided into two steps,in the first step,there were two proteins that SHPG and PZP showed the abundance different in T1 D from healthy,while another three proteins that CRP,CETP and SERPIND1 exhibited the different in T2 D from healthy.The five proteins were thus removed from the candidate list,and the rest 7 proteins were taken for further examination of the MODY specificity.In the comparison of the paired group,the abundances of all the 7 proteins,SERPINA1,SERPINA7,APOC4,LPA,CD14,C6,and F5,in MODY were significantly different from either T2 D or healthy,while of the five proteins,SERPINA7,APOC4,LPA,C6 and F5,in MODY were obviously distinct from T1 D.Taking all the statistical comparisons together,the five proteins,SERPINA7,APOC4,LPA,C6 and F5,were not only found the abundance changes in MODY against the correspondent abundances in the family members with normal blood glucose levels,but were also perceived the significant alternations in MODY from that in T1 D and T2 D.The protein signature consisting of SERPINA7,APOC4,LPA,C6 and F5 was therefore proposed as the MODY indicator,at least in the MODY family.Conclusion: Obvious,the quantitative proteomics in this study discover 5 new serum proteins as diabetic indicator,and the protein signature to indicate MODY,especially for the Uyghur MODY family,indeed opens a new path to develop the serum indicators related with diabetes and to take an overview to the responses of serum components in diabetes.The application of this protein signature to MODY is still questionable whether it is accommodated in all the MODY sera.We are working toward the direction to further verify the rationality of the protein signature defined by this study in many diabetes serum samples.