Reproducibility and reliability of protein quantity analyzed by DIA quantitative proteomics for urine biomarker discovery

Introduction: Currently, quantitative proteomics has improved its accuracy and sensitivity by using highly advanced mass spectrometry and analysis tools, enabling comprehensive quantification of proteomes. In our laboratory urine biomarkers for diseases have been searched by proteomics. In general, quantification of highly abundant proteins is reliable however, biomarker proteins may be trace in urine and quantification of these proteins were not reliable. In addition, plasma proteins are often massive in urine, especially in kidney disease patient urine, making discovery of biomarkers of trace quantities difficult. In this study we investigated the dynamic range of quantities of urinary proteins by recently advanced technologies of quantitative proteomics. Furthermore, we challenged to find biomarkers among the low-abundant proteins.

Methods: Proteins were extracted from a mixture of urine from six healthy volunteers (HV) or six diabetic patients (DM) by methanol/chloroform precipitation. After digestion with trypsin, the peptides were purified with C-18 column and 200 ng each were measured 5 times by Bruker's TimsTOFpro with DIA mode. Then quantitative analysis was performed with DIA-NN analysis software. After normalization of peptides, peptide quantity was estimated after multipling by 10^6 (ppm). Then protein amounts were calculated by summing the peptides quantities (ppm), which were assigned to a given protein. The reproducibility of the quantification for each protein across runs was evaluated by a coefficient of variation (CV). In addition, parallel fluctuating changes of quantities of the peptides assigned to a protein also demonstrated the reliability of the quantification of each protein.

Results: The quantitative data of approximately 3,000 urinary proteins were obtained with the dynamic range from 10^4 to 10^-1ppm. The most abundant protein in urine was serum albumin; 2.24x10^4ppm in HV and 3.25x10^4ppm in DM. The CV of albumin quantities (ppm) was 4.9％ in HV and 3.1％ in DM. The CVs of top 1,000 highly abundant proteins (10^3~10^5ppm), approximately one-third of all proteins, were less than 20%. Interestingly, more than half of the proteins of below 10 ppm, which accounted for about 20% of the total, had CVs of 20% or less, indicating that the quantitative values of even very trace amounts of proteins are also accurate. For proteins with high CVs, we examined whether the quantitative values of multiple peptides detected as those assigned to a given protein were fluctuated synchronously, and examined the cause of the high CVs. The analysis suggested that one of the reasons of the high CVs may be mismatches of peptides, which were shown by unsynchronized fluctuation of the quantities of a few peptides assigned to a given protein among different urine samples.