Development and Application of Multiplexed Proteomics to Investigate Protein Complexes in Kidney Aging and Disease
- Glomerular Diseases: From Inflammation to Fibrosis - II
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: From Inflammation to Fibrosis
- Nguyen, Thao, Calico Life Sciences LLC, South San Francisco, California, United States
- Seitzer, Phillip, Calico Life Sciences LLC, South San Francisco, California, United States
- Hirohama, Daigoro, University of Pennsylvania, Philadelphia, Pennsylvania, United States
- Olsson, Niclas, Calico Life Sciences LLC, South San Francisco, California, United States
- Chan, Leanne, Calico Life Sciences LLC, South San Francisco, California, United States
- Abedini, Amin, University of Pennsylvania, Philadelphia, Pennsylvania, United States
- Susztak, Katalin, University of Pennsylvania, Philadelphia, Pennsylvania, United States
- Mcallister, Fiona E., Calico Life Sciences LLC, South San Francisco, California, United States
Kidney aging is a complex process associated with a decline in renal function. This decline is due to several factors, including changes in cellular states driven by protein expression and their composition in complexes. Proteins rarely function alone, and their activity is often determined by their interactions with others. However, owing to the technical challenges, no attempt has been made to profile the global changes in protein complexes composition and abundance in the kidney as a function of age or disease.
Co-fractionation mass spectrometry (CF-MS) is an emerging unbiased method that identifies protein complexes in cell or tissue lysate. CF-MS first separates the proteins in a sample by chemical properties such as size and charge using native chromatography, and thereby proteins from the same complexes will co-elute into the same fractions. All the proteins in each fraction are then identified and quantified by MS and the complexes can be inferred using downstream bioinformatics analysis. In a classical CF-MS set-up, one fraction per sample is processed and analyzed by the MS at a time. As samples are fractionated into tens or hundreds of fractions, with each fraction requiring analysis, the large number of samples results in a highly time-consuming process both in sample preparation as well as data acquisition time.
To address this bottleneck, we have explored using a multiplexed isobaric labeling strategy (Tandem Mass Tags, TMT) that can significantly reduce the acquisition time. We have applied this approach to analyze young and old human kidneys as well as kidneys from chronic kidney disease patients.
We present the first comparison of profiling global changes in protein complexes in young vs old vs CKD human kidneys. We validate our novel TMT proteomics approach using known complexes before applying the technique to profile complexes in precious human kidneys. Our preliminary results suggest that the method is a promising high throughput tool for comprehensively studying protein complexes in kidney aging and disease.
Our approach of using CF-MS combined with TMT-MS offers a novel direction to explore changes in protein complexes with a great potential to identify new drug targets and therapies for kidney aging and diseases.