Immuno affinity purification combined with mass spectrometry detection for the monitoring of VEGF isoforms in patient tumor tissue

Abstract

Vascular endothelial growth factor (VEGF) is a key mediator of tumor growth and metastasis. At least 7 isoforms of the protein exist due to differential splicing, but little is known about the biological activities of these different isoforms. We purified VEGF from resected human bronchial carcinoma tissue and normal bronchus by immuno affinity chromatography (IAC) and immuno capture (IC) on a ProteinChip® Array, identifying the isoforms by MS. Both methods proved to be useful for the analysis of protein isoforms, which are present in situ in tumor tissue. Different expression patterns of VEGF were demonstrated in tumor tissue compared to healthy tissue. The possibility to analyse VEGF protein isoforms in situ creates new opportunities to study the regulation of angiogenesis in healthy and malignant tissues.

Development of CE methods to analyze potential components of the angiogenic glycoprotein vascular endothelial growth factor 165

“…It is known by LC-MS and RP-HPLC of the tryptic digest that human VEGF 165 expressed in Chinese hamster ovary cells can exist in non-glycosylated and glycosylated forms at Asn 75, and that the biantennary carbohydrate structure exists in the non-sialylated, monosialylated, and disialylated forms [52]. Based on the amino acid sequence several O-linked glycosylations are possible and variations found by MS in VEGF purified from human bronchial tissue could be attributable to this type of glycosylation [55]. However, for VEGF extracted from guinea pig line 10 tumor cells, the existence of N-glycosylation and the absence of O-glycosylation have been proved [56].…”

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“…Although recent studies 24 have shown that the quantity of VEGF-A expressed by tumor cells affect clinical outcome, none of the available techniques employed in quantifying tumor VEGF-A in solid tumors are routine and therefore not certain if such techniques were widely undertaken that they would be clinically useful or cost-effective to predict outcomes in individual patients 18 .…”

A new strategy of studying protein–protein interactions: Integrated strong anion exchange/reversed‐phase chromatography/immunoprecipitation coupled with mass spectrometry for large‐scale identification of proteins interact with immunoglobulin G in HeLa cells

“…The experimental methods to determine PPIs can be divided into the following two major classes: (1) Binary methods: These methods are designed to test each specific interaction between a pair of proteins, such as yeast two‐hybrid system [4], mammalian split‐luciferase system [5], and fluorescence resonance energy transfer [6]. (2) Co‐complex methods: In these methods, one tagged specific protein (bait) is used, which interrogates its interaction with a group of proteins (prey) in order to find direct and indirect physical associations, such as immunoprecipitation (IP) [7], affinity purification–MS [8], and cross‐linking MS [9]. Despite considerable success in studying PPIs based on existing techniques, a lot more systematic explorations are still urgently demanded for large‐scale identification of PPIs.…”