The embodiments herein provide methods to analyze the in vitro stability of recalcitrant or membrane-bound proteins in simulated gastric fluid (SGF) comprising the proteolytic enzyme, pepsin, and in combination with a novel pepsin-trypsin assay employing state-of-the-art mass spectrometric approaches, such as LC-MS/MS, to monitor the precise degradation products. The extent of protein digestion can be evaluated by the appearance of peptic products and the disappearance of tryptic peptide products (as a proxy for intact protein). The embodiments herein also provide methods for protein quantitation using high-sensitivity LC-MRM-MS quantification. The methods embodied herein are particularly useful in charactering proteins produced in transgenic plants, such as canola genetically engineered to produce long chain omega-3 polyunsaturated fatty acids.

Methods and kits for preparing liquid samples are presently claimed and described. The method may include treating liquid sample with a hydrolysis enzyme, hydrolyzing the liquid sample to prepare a hydrolysate, and purifying the hydrolysate with magnetic based purification. In certain aspects, the hydrolysis enzyme is bound to a magnetic bead or a magnetic particle. Kits for preparing a liquid sample can include a hydrolysis enzyme, magnetic beads or magnetic particles, one or more internal standards, a liquid chromatography column and one or more solvents to be used as mobile phases, one or more calibrant solutions and instructions for use.

A pretreatment method for detection of an undenatured type II collagen in a cartilage collagen product or a cartilage is proposed. The method can separate an undenatured type II collagen from a complex environment system including proteoglycan, a hydrolyzed collagen, open-chain denatured collagen and the like, and present a dissolved state, thereby facilitating subsequent further qualitative and quantitative detection.

A method is provided for evaluating the suitability of a duodenal fluid sample collected from an animal as a sample for detecting pancreatic fluid-derived components. The method includes: (a) mixing a duodenal fluid sample with a chymotrypsin-specific substrate and measuring an amount of degradation the chymotrypsin-specific substrate by of the duodenal fluid sample, (b) mixing the duodenal fluid sample with a pepsin-specific substrate and measuring an amount of degradation of the pepsin-specific substrate by the duodenal fluid sample, and (c) evaluating that the duodenal fluid sample is suitable as a sample for detecting pancreatic fluid-derived components if the amount of degradation of the chymotrypsin-specific substrate by the duodenal fluid sample is higher than a prescribed threshold value and the amount of degradation of the pepsin-specific substrate by the duodenal fluid sample is lower than a prescribed threshold value, as being suitable as a sample for detecting pancreatic fluid-derived components.

A method is provided for evaluating the suitability of a duodenal fluid sample collected from an animal as a sample for detecting pancreatic fluid-derived components. The method comprises: (a) mixing a duodenal fluid sample with a chymotrypsin-specific substrate and measuring an amount of degradation the chymotrypsin-specific substrate by of the duodenal fluid sample, (b) mixing the duodenal fluid sample with a pepsin-specific substrate and measuring an amount of degradation of the pepsin-specific substrate by the duodenal fluid sample, and (c) evaluating that the duodenal fluid sample is suitable as a sample for detecting pancreatic fluid-derived components if the amount of degradation of the chymotrypsin-specific substrate by the duodenal fluid sample is higher than a prescribed threshold value and the amount of degradation of the pepsin-specific substrate by the duodenal fluid sample is lower than a prescribed threshold value, as being suitable as a sample for detecting pancreatic fluid-derived components.

The embodiments herein provide methods to analyze the in vitro stability of recalcitrant or membrane-bound proteins in simulated gastric fluid (SGF) comprising the proteolytic enzyme, pepsin, and in combination with a novel pepsin-trypsin assay employing state-of-the-art mass spectrometric approaches, such as LC-MS/MS, to monitor the precise degradation products. The extent of protein digestion can be evaluated by the appearance of peptic products and the disappearance of tryptic peptide products (as a proxy for intact protein). The embodiments herein also provide methods for protein quantitation using high-sensitivity LC-MRM-MS quantification. The methods embodied herein are particularly useful in charactering proteins produced in transgenic plants, such as canola genetically engineered to produce long chain omega-3 polyunsaturated fatty acids.

Provided herein are methods, kits and compositions for detecting an analyte of interest to interrogate spatial gene expression in a sample using templated ligation.