ABSTRACT: The LC-MS study was carried out to identify the proteins in urine samples of clinical cases of diabetic nephropathy (Healthy control, Diabetic group, Microalbuminuria, Overt albuminuria group).
Study design
The urine samples (10 ml) of patients with HbA1c greater than 6.5 percent were collected in a sterile container from the endocrinology OPD, PGIMER, Chandigarh, India, after obtaining informed consent. The study was carried out following the Helsinki Declaration. The Institute Ethics Committee approved the study protocol via (IEC) vide No INT/IEC/2020/SPL-1031.
The spot dipstick method was used to screen urine samples for the presence of blood (haematuria) and pus cells (leucocytes). Samples showing the presence of both blood and leucocytes were further excluded from the study. After screening, 90 patient samples (30 diabetic controls, 30 microalbuminuria, and 30 overt albuminuria) were included in the study. For control, urine samples of 30 healthy controls were also included in the study.
The albumin excretion rate parameters (Albumin to creatinine ratio (ACR)) was measured in Alere Afinion TM AS100 Analyser with ACR test kits. The ACR was expressed in mg per gram. Based on the obtained ACR, the patient groups were categorized following criteria as detailed under inclusion criteria. The inclusion and exclusion criteria are mentioned below.
Inclusion criteria
Microalbuminuria (meant as diabetic microalbuminuria)- Established case of Diabetes Mellitus with glycated hemoglobin more than or equal to 6.5 percent and urinary albumin excretion rate 30 to 300 mg per gram of creatinine in a spot urine sample.
Overt albuminuria (meant as diabetic overt albuminuria): Established case of Diabetes Mellitus with glycated hemoglobin more than or equal to 6.5 percent and urinary albumin concentration more than or equal to 300 mg per gram of creatinine in a spot urine sample.
Diabetic Control- Established case of diabetes mellitus with glycated hemoglobin more than or equal to 6.5 percent and urinary albumin excretion rate of less than 30 mg per gram of creatinine in a spot urine.
Exclusion criteria
Patients with any evidence of urine tract infection or haematuria by dipstick test.
Separation of urinary proteins
The urinary proteins are separated from the urine matrix through centrifugation (15000 g for 1 hour at 4 degrees Celsius. The pellet obtained was expected to contain the proteins. For this, a 2 ml urine sample was subjected to centrifugation for each participant. After centrifugation (1ml in different aliquots), 2 pellets from the same sample were obtained. The pellets were suspended in PB (200 microlitres, pH 7.38, 0.1 M) and mixed well with a vortex. The pellets obtained from the same group were pooled. A similar treatment was provided to all the samples to obtain pellets. Protein quantification was done using the BCA method.
LC-MS protocol: For this, 100 micrograms of urinary protein in 10 mM Tris HCl buffer, pH 7.38 was denatured by boiling with 10 mM dithiothreitol for 10 mins followed by the addition of Iodoacetamide (90 mM, SIGMA) and incubation for 30 mins in the dark at 37 degrees Celsius. These were then subjected to trypsin digestion (2 micrograms, Promega, MS grade, prepared in acetic acid) and kept overnight at 37 degrees Celsius. The reaction was stopped by the addition of 1 percent trifluoroacetic acid (TCI, LC-MS grade). The sample clean-up with C18 resins column was performed and the aliquots were then sent for data acquisition (PMID 39216816).
The different raw files obtained are named as follows and uploaded.
1- Healthy Control
2- Diabetic Controls
3- Microalbuminuria
4- Overt albuminuria
Prof Dibyajyoti Banerjee, Dr. Rajasri Bhattacharyya, Dr. Sheemona Chowdhary, Dr. Deepak Kumar, and Ms Monu Kumari acknowledge ICMR, New Delhi, India, sanctioned project titled Pseudoesterase activity of albumin as a determinant for serum cholesterol in rabbits for financial support.
Dibyajyoti Banerjee 
