Session: Session 2
Toward rapid and robust proteomics analysis of urine from patients cohorts
Urine is a biological fluid of prime importance in studies of kidney diseases: it is readily available and non-invasive. However, the proteomic analysis of urine samples is challenging due to the high variability in urine concentration and composition. Currently, a preliminary step of protein precipitation is usually required prior to mass spectrometry analysis, leading to longer protocol and further variability. In this study, we present a new workflow for high throughput and in-depth direct proteomic analysis of urine samples that doesn’t require the precipitation step.
Urine samples were collected from five different healthy patients for optimization. One aliquot of each sample (1mL) was subjected to TCA precipitation prior to the digestion, and two other aliquots (150µL and 100µL) were directly digested on a Strap plate (Protifi™). Then, 600ng of peptides were injected into an EvosepOne LC-TimsTOF HT MS (Bruker Daltonics) with a 30 Samples Per Day (SPD) or a 60 SPD gradient. Mass spectrometry data were acquired using diaPASEF mode and analysed by DIANN v1.8.1.
We compared results obtained after TCA precipitation, direct digestion on 150µL and 100µL of urine. We could identify 12% more proteins after TCA precipitation (4135±585 proteins) than after direct digestion on 100µL of urine (3737±527 proteins) and 150µL (3676±645 proteins). However, we found 55 proteins enriched in the direct digestion experiment compared to the TCA, suggesting an enrichment of a new set of proteins which may have been lost after precipitation. We also the impact of a longer LC gradient leading to 4769±601 proteins for TCA precipitation, 4382±561 proteins for 100µL of urine.
A cohort of over 160 patients affected by rare genetic diseases are currently being processed and compared to the TCA precipitation protocol already analysed.
This study provides a simple and direct protocol for urine samples preparation allowing large scale proteomics analysis starting from low volumes of urine.
Urine samples were collected from five different healthy patients for optimization. One aliquot of each sample (1mL) was subjected to TCA precipitation prior to the digestion, and two other aliquots (150µL and 100µL) were directly digested on a Strap plate (Protifi™). Then, 600ng of peptides were injected into an EvosepOne LC-TimsTOF HT MS (Bruker Daltonics) with a 30 Samples Per Day (SPD) or a 60 SPD gradient. Mass spectrometry data were acquired using diaPASEF mode and analysed by DIANN v1.8.1.
We compared results obtained after TCA precipitation, direct digestion on 150µL and 100µL of urine. We could identify 12% more proteins after TCA precipitation (4135±585 proteins) than after direct digestion on 100µL of urine (3737±527 proteins) and 150µL (3676±645 proteins). However, we found 55 proteins enriched in the direct digestion experiment compared to the TCA, suggesting an enrichment of a new set of proteins which may have been lost after precipitation. We also the impact of a longer LC gradient leading to 4769±601 proteins for TCA precipitation, 4382±561 proteins for 100µL of urine.
A cohort of over 160 patients affected by rare genetic diseases are currently being processed and compared to the TCA precipitation protocol already analysed.
This study provides a simple and direct protocol for urine samples preparation allowing large scale proteomics analysis starting from low volumes of urine.