Comprehensive proteogenomic characterization of rare kidney tumors.

Non-clear cell renal cell carcinomas (non-ccRCCs) encompass diverse malignant and benign tumors. Refinement of differential diagnosis biomarkers, markers for early prognosis of aggressive disease, and therapeutic targets to complement immunotherapy are current clinical needs. Multi-omics analyses of 48 non-ccRCCs compared with 103 ccRCCs reveal proteogenomic, phosphorylation, glycosylation, and metabolic aberrations in RCC subtypes.

Developing quantitative assays for six urinary glycoproteins using parallel reaction monitoring, data-independent acquisition, and TMT-based data-dependent acquisition.

Quantitative approaches encompassing parallel reaction monitoring (PRM), data-independent acquisition (DIA), and data-dependent acquisition (DDA) are commonly used to investigate protein expression profiles. However, analytical performances of assays developed using PRM, DIA, and Tandem Mass Tag (TMT)-based DDA for quantitative proteomics have yet not been investigated. Here, we developed assays for glycopeptides identified from six glycoproteins, including Leucine-rich alpha-2-glycoprotein (LRG1), Prostaglandin-H2 D-isomerase (PTGDS), Aminopeptidase N (ANPEP), CD63 antigen (CD63), Clusterin (CLU), and Prostatic acid phosphatase (ACPP), using PRM, DDA, and DIA and evaluated the analytical performances of each assay using the different acquisition modes.

Development of Parallel Reaction Monitoring Assays for the Detection of Aggressive Prostate Cancer Using Urinary Glycoproteins.

Recently, we have found that two urinary glycoproteins, prostatic acid phosphatase (ACPP) and clusterin (CLU), combined with serum prostate-specific antigen (PSA) can serve as a three-signature panel for detecting aggressive prostate cancer (PCa) based on a quantitative glycoproteomic study. To facilitate the translation of candidates into clinically applicable tests, robust and accurate targeted parallel reaction monitoring (PRM) assays that can be widely adopted in multiple labs were developed in this study. The developed PRM assays for the urinary glycopeptides, FLN*ESYK from ACPP and EDALN*ETR from CLU, demonstrated good repeatability and a sufficient working range covering three to four orders of magnitude, and their performance in differentiating aggressive PCa was assessed by the quantitative analysis of urine specimens collected from 69 nonaggressive (Gleason score = 6) and 73 aggressive (Gleason ≥ 8) PCa patients.

Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.

Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-tip using the standard protein bovine fetuin.

Combinatorial genome and protein engineering yields monoclonal antibodies with hypergalactosylation from CHO cells.

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities.

Isoform separation of proteins by mixed-mode chromatography.

Mixed-mode chromatography uses a multimodal functional resin, mainly composed of electrostatic and aromatic/hydrophobic groups. Here we have tested 2 mixed-mode resins, anion-exchange Capto adhere and cation-exchange Capto MMC, using 2 model proteins, i.e.