Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients

Abstract

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent “top-down” proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight–based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum-rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.

Discovery and validation of a novel blood-based molecular biomarker of rejection following liver transplantation

“…Prior studies have demonstrated genetic polymorphisms, gene expression profiles, blood lymphocyte populations, and complement proteins associated with AR in the LT population. [15][16][17][18][20][21][22][23][24]28,[55][56][57][58] The major limitation of these studies has been small sample sizes, lack of validation cohorts, and statistical rigor and biomarker anal- As part of the CTOT-14 study, we have also recently reported a novel clinical/protein model (PRESERVE) that can predict deterioration in renal function early after LT. 60 In future trials, we envision evaluating both tests to select patients for early CNI minimization/ withdrawal (renal deterioration predicted by PRESERVE) and serially for more optimal decision-making at each time point-for example, continue minimization if the blood test is below the threshold (eg, "TX") vs stopping minimization if above the threshold (eg, "AR").…”

“…Finally, the lack of progress in this area in LT may be related to difficulties inherent to the development of predictive biomarkers. Prior studies have demonstrated genetic polymorphisms, gene expression profiles, blood lymphocyte populations, and complement proteins associated with AR in the LT population 15‐18,20‐24,28,55‐58 . The major limitation of these studies has been small sample sizes, lack of validation cohorts, and statistical rigor and biomarker analysis mostly performed only at diagnosis.…”

Genomics and Liver Transplantation: Genomic Biomarkers for the Diagnosis of Acute Cellular Rejection

“…Mapping the analysis of identified proteins back to genes revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. (84) MetABolites Metabolites are extremely sensitive to different biological states and, as such, may represent a promising approach to better understand the biopathology underlying ACR. Most studies have focused on metabolic pathways and individual metabolites modulating immune cell function and immune responses, but there are a few studies focusing exclusively on altered metabolic pathways associated with ACR.…”

Serum proteomic predicts effectiveness and reveals potential biomarkers for complications in liver transplant patients

“…Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in protein analysis for rapid and accurate identification of biomarkers and mechanisms [5]. While various cancer biomarkers have been explored [6], only a few studies have focused on non-cancer diseases [7][8][9][10] and a little progress is reported on biomarker discovery in solid organ transplantations [11][12][13][14][15]. Proteomics provides a reliable and effective tool to identify disease markers that can help to evaluate the illness state of patients in liver transplantation.…”