Enzymatic Digestion of Proteins in Biological Samples for Quantification with LC-MS/MS

Willems, Suzanne (2021) Enzymatic Digestion of Proteins in Biological Samples for Quantification with LC-MS/MS. Bachelor's Project, Pharmacy.

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Abstract

The enzymatic digestion of proteins in biological samples is an important step for the quantification with LC-MS/MS and is presented here in an overview. The enzymes used for this are classified as proteases and work by the hydrolysis of the peptide bonds. The most commonly used enzymes are trypsin, chymotrypsin, Glu-C, Lys-C, Arg-C, Asp-N and pepsin, each with their own cleavage sites. The digestion step can be optimized by adjusting several factors: the pH, the temperature, the enzyme:protein ratio and the way the enzyme is used. An evaluation of these enzymes was performed by theoretically digesting hGH 1 (22 kDa) and showed that trypsin resulted in peptides with an optimal length (7-20 amino acids) and charge (at least two positive charges) and had a relatively low price with a wide availability. In general, all the enzymes produced peptides with SSRC (hydrophobicity) scores that lie around the useful range of 10 to 45. Furthermore, a demonstration of signature peptide selection was performed after trypsin digestion on two isoforms of hGH with a mass of 22 kDa by taking several criteria into account: uniqueness and the absence of unstable amino acids. This resulted in two unique peptides for hGH 1 and three for hGH 2. Lastly, the importance of the use of internal standards is discussed. Stable-isotope labeled (SIL) forms of the protein cover for all the steps in the digestion, but may be hard to obtain; SIL forms of the signature peptide often are a good alternative. Next

Item Type:	Thesis (Bachelor's Project)
Supervisor name:	Sleumer, B. and Merbel, N.C. van de
Degree programme:	Pharmacy
Thesis type:	Bachelor's Project
Language:	English
Date Deposited:	07 Apr 2021 15:13
Last Modified:	07 Apr 2021 15:13
URI:	https://fse.studenttheses.ub.rug.nl/id/eprint/24161

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