Proline and hydroxyproline are essential amino acids that play a crucial role in collagen synthesis in mice. Collagen is the most abundant protein in the body and provides structural and functional support for various tissues, such as skin, bone, cartilage, and blood vessels. The concentration of proline amino acid and hydroxyproline in biological samples, such as blood, urine, or tissue, can reflect the status of collagen metabolism and turnover and can be used as indicators of physiological and pathological conditions, such as aging, inflammation, and cancer. However, the analysis of proline amino acid and hydroxyproline in mouse tissues poses several analytical challenges, such as the low concentration of these amino acids in the tissue matrix, the high polarity of these amino acids that makes them difficult to retain and separate by conventional reversed-phase chromatography, the interference from other amino acids and peptide-bound forms of proline and hydroxyproline that can affect the accuracy and specificity of the measurement, and the presence of different endogenous levels of proline and hydroxyproline in various tissues, such as liver, kidney, lung, and heart, that adds to the complexity of accurate quantification. Therefore, a sensitive, selective, and reliable method is needed to quantify proline and hydroxyproline in mouse tissues, which can help to elucidate the role and regulation of these amino acids in collagen synthesis and degradation and their involvement in various disease processes. Here, hydrophilic interaction liquid chromatography (HILIC) offers a promising approach due to its compatibility with polar analytes like proline amino acids.
To address these challenges, we developed and validated a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for quantitating proline amino acids and hydroxyproline in mouse tissues. The method involved the following steps: (1) tissue homogenization and acid hydrolysis of the tissue, which released the free amino acids from the tissue matrix and the peptide-bound forms of proline and hydroxyproline; (2) a time-course study, which determined the optimal duration of hydrolysis for each tissue type, based on the maximum recovery of proline amino acid and hydroxyproline; (3) protein precipitation with acetonitrile, which removed the proteins and other interfering substances from the hydrolysate; (4) dilution of the supernatant with water, which adjusted the solvent composition and the concentration of the analytes for injection into the LC–MS/MS system; (5) chromatographic separation on a hydrophilic interaction liquid chromatography (HILIC) column, which retained and separated the polar amino acids by using a gradient elution of acetonitrile and water with 0.1% formic acid; (6) detection and quantification of proline amino acid and hydroxyproline by a mass spectrometer, which operated in positive electrospray ionisation mode and multiple reaction monitoring mode, which selected and measured the specific precursor and product ions for each analyte; (7) use of a mix of amino acids as internal standard, which compensated for the matrix effect and the variability in the sample preparation and analysis, and which provided a unique internal standard that accurately reflected the changes in both analytes concentration and exhibited a linear response with the concentration of proline amino acid and hydroxyproline. Thus, our comprehensive approach ensures precise and reliable quantification in the proline assay, overcoming the challenges of analyzing these amino acids in mouse tissues. It is hypothesised that disruptions in collagen synthesis, often marked by changes in proline amino acid and hydroxyproline metabolism, are associated with debilitating conditions, including fibrosis and connective tissue disorders. With our method’s precision in measuring these amino acids, researchers can delve deeper into understanding the underlying mechanisms of collagen-related diseases and explore potential therapeutic interventions to modulate collagen synthesis pathways to alleviate human suffering.
The method was validated according to the US Food and Drug Administration guidelines for bioanalytical methods and demonstrated good accuracy, precision, linearity, selectivity, recovery, matrix effect, and stability for measuring proline amino acid and hydroxyproline in mouse tissues, crucial components for collagen synthesis. The method utilized hydrophilic interaction liquid chromatography (HILIC) as a robust separation technique. This chromatographic approach provided excellent resolution and peak shape for proline and hydroxyproline, facilitating accurate quantification.
The technique was used to study how free amino acid proline and hydroxyproline alter in different mouse lung tissues, such as normal, inflamed, fibrotic and tumor tissues, by hydrophilic interaction liquid chromatography for good compound separation. The chromatographic technique was our main tool for detecting proline amino acid and hydroxyproline at micromolar levels. HILIC sensitivity and applicability for analyzing these analytes in complex bio-matrices were also demonstrated.
The method revealed significant differences in the concentration and ratio of proline and hydroxyproline among different tissue types, which indicated the changes in collagen metabolism and turnover in different disease conditions. The proline assay method could help study the role and regulation of proline amino acid and hydroxyproline in mouse models of various diseases, such as inflammation, fibrosis, and cancer, and evaluate the effect of therapeutic interventions on collagen synthesis and degradation. The proline assay method could also be extended to other tissue types, such as liver, kidney, and heart, and to other biological samples, such as blood and urine, to assess the exposure and risk of proline amino acid and hydroxyproline in mouse populations.