Quantitative Proteomics Based on Optimized Data-Independent Acquisition in Plasma Analysis

The advent of high-resolution and frequency mass spectrometry has ushered in an era of data-independent acquisition (DIA). This approach affords enormous multiplexing capacity and is particularly suitable for clinical biomarker studies. However, DIA-based quantification of clinical plasma samples is a daunting task due to the high complexity of clinical plasma samples, the diversity of peptides within the samples, and the high biologic dynamic range of plasma proteins. Here we applied DIA methodology, including a highly reproducible sample preparation and LC–MS/MS analysis, and assessed its utility for clinical plasma biomarker detection. A pancreatic cancer-relevant plasma spectral library was constructed consisting of over 14 000 confidently identified peptides derived from over 2300 plasma proteins. Using a nonhuman protein as the internal standard, various empirical parameters were explored to maximize the reliability and reproducibility of the DIA quantification. The DIA parameters were optimized based on the quantification cycle times and fragmentation profile complexity. Higher analytical and biological reproducibility was recorded for the tryptic peptides without labile residues and missed cleavages. Quantification reliability was developed for the peptides identified within a consistent retention time and signal intensity. Linear analytical dynamic range and the lower limit of quantification were assessed, suggesting the critical role of sample complexity in optimizing DIA settings. Technical validation of the assay using a cohort of clinical plasma indicated the robustness and unique advantage for targeted analysis of clinical plasma samples in the context of biomarker development

GAIP Interacting Protein C-Terminus Regulates Autophagy and Exosome Biogenesis of Pancreatic Cancer through Metabolic Pathways

<div><p>GAIP interacting protein C terminus (GIPC) is known to play an important role in a variety of physiological and disease states. In the present study, we have identified a novel role for GIPC as a master regulator of autophagy and the exocytotic pathways in cancer. We show that depletion of GIPC-induced autophagy in pancreatic cancer cells, as evident from the upregulation of the autophagy marker LC3II. We further report that GIPC regulates cellular trafficking pathways by modulating the secretion, biogenesis, and molecular composition of exosomes. We also identified the involvement of GIPC on metabolic stress pathways regulating autophagy and microvesicular shedding, and observed that GIPC status determines the loading of cellular cargo in the exosome. Furthermore, we have shown the overexpression of the drug resistance gene ABCG2 in exosomes from GIPC-depleted pancreatic cancer cells. We also demonstrated that depletion of GIPC from cancer cells sensitized them to gemcitabine treatment, an avenue that can be explored as a potential therapeutic strategy to overcome drug resistance in cancer.</p></div

GIPC induce autophagy in the pancreatic cancer cells.

<p>A) AsPC-1 and PANC-1 cells were infected with lentiviruses expressing shRNAs to GIPC and scrambled control. An equal amount of whole-cell lysates from AsPC-1 and PANC-1 GIPC depleted cells were analyzed by immunoblotting (IB) with the antibodies for GIPC and LC3II. β-Actin is used as loading control. B) A representative immunofluorescence analysis of PANC-1 cells for expression of LC3 II (green) in GIPC depleted PANC-1 cells compared to the control cells. Cells were counterstained with DAPI (blue).</p

GIPC modulates expression of drug resistance associated gene <i>ABCG2</i> and sensitizes pancreatic cancer cell lines to gemcitabine.

<p>A) ABCG2 expression was confirmed at protein level by western blot in GIPC knockdown and control cells as well as in corresponding exosomes. PLC γ is used as loading control for exosomes and β-Actin is used as loading control for cell lysates. B) GIPC +/- PANC-1 cells were treated with different concentration of the gemcitabine for 72 h. Effect of the drug treatment was evaluated using MTS cell viability assay. The horizontal bar represents the IC50 level.</p

GIPC regulates autophagy by interfering with glucose uptake.

<p>A) Quantitative PCR and B) Western blot analysis of Glut1 to analyze the effect of GIPC-depletion in Glut1 expression. β-Actin is used as loading control. Both Glut1 mRNA and protein levels decreased significantly upon GIPC depletion in AsPC-1 and PANC-1 cells. C) Glucose uptake was significantly decreased in GIPC depleted cells as compared to the control cells in AsPC-1 and PANC-1 cells (** denotes p<0.01). D) Intracellular glucose levels were also significantly decreased in the GIPC depleted AsPC-1 and PANC-1 cell lines confirming the role GIPC in glucose metabolism (** denotes p<0.01).</p

GIPC increase exosome secretion and biogenesis.

<p>Exosomes were isolated from culture media of AsPC-1 and PANC-1 GIPC depleted and control cell culture. For qualitative measurement same amount of cells were seeded in culture plates. A) A comparison of activity of acetylcholine esterase is depicted for exosomes collected from GIPC depleted and control AsPC-1 cell line. B&C) A comparison of total RNA content of the exosome preparation from AsPC-1 and PANC-1 cell culture is displayed. D) A representative size distribution profile of the exosome preparation is obtained using Nanosight. E) A representative transmission electron micrograph (TEM) of exosomes is presented in this figure. Scale bar is 500 nm. F) A higher magnification TEM image of exosomes is presented. The scale bar is 200 nm. G) Immunoblot conducted with cells lysates collected from GIPC knockdown cells as well as control cells were being probed with TSG 101, Alix, and CHMP 4B. PLC γ is used as loading control.</p

GIPC regulates stress induced metabolic pathways.

<p>A) Immunoblot of the cell lysates from GIPC depleted and control AsPC-1 and PANC-1 cells are being probed with p-AMPK-α, total AMPK-α. β-Actin is used as loading control. B) Further, immunoblot of cell lysates from above condition were being probed with p–mTOR, total mTOR, p-p70S6K and total p70S6K. β-Actin is used as loading control.</p

The role of Beclin 1 and Atg 7 in GIPC induced autophagy.

<p>A) Immunoblot analysis of the Atg7 expression in AsPC-1 and PANC-1 cell lysates transfected with siRNA to GIPC, Atg7 and scrambled control. β-Actin is used as loading control. B) Immunoblot analysis of the Beclin1 expression in AsPC-1 and PANC-1 cell lysates transfected with siRNA to GIPC, Beclin1, and scrambled control. β-Actin is used as loading control. C) Co-immunoprecipitation (IP) of the PANC-1 cell lysates transfected with siRNA to GIPC, and scrambled control using GIPC antibody. Immunocomplexes were analyzed by immunoblotting (IB) with antibodies to Beclin1 and GIPC.</p