1. Collagen is a major protein in connective tissue and the extracellular matrix
ID: 198907 • Letter: 1
Question
1. Collagen is a major protein in connective tissue and the extracellular matrix. Both the Bradford assay and absorbance at 280 nm cannot be used to determine the concentration of collagen. Why is this?
2. The old way of determining the amino acid composition of a protein involved hydrolyzing the peptide bonds joining the amino acids, separating the amino acids by chromatography, then determining the relative amounts of each.How would a modern scientist determine the amino acid composition of a protein?
Explanation / Answer
Answer:
1. Bradford dye can bind to these amino acids in protein sample - arginine (Arg) and phenylalanine (Phe), tryptophan (Trp), and proline (Pro) (aromatic amino acid residues).
Tryptophan, tyrosine and cysteine are extremely rare in collagen, with the result that collagen is virtually invisible in terms of 280nm absorbance and also does not give the desired color with bradford.
2. In modern times, mass spectrometry (MS) is widely used in the characterization of proteins at the intact (native) and peptide levels, as well as in the analysis of enzymatically generated protein fragments, in conjunction with an expanding range of compatible chromatographic separation techniques.
Electrospray ionization of intact proteins generates a charged envelope of peaks corresponding to differing degrees of ionization that can readily be converted to the molecular mass using a mathematical algorithm. Typically, average mass is calculated; however, high mass accuracy time-of-flight and Fourier transform instruments allow isotopic resolution, which can be important in the study of protein fragments to determine sequence and post-translational modifications (PTMs).
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