Optimizacija priprave proteinov krvne plazme z razgradnjo na peptide v raztopini za proteomsko analizo z masno spektrometrijo

diplomsko delo univerzitetnega študijskega programa I. stopnje

Eva Zajšek (Author), Uroš Potočnik (Mentor), Maya Petek (Co-mentor)

Abstract

V diplomskem delu sta predstavljena danes najpogostejša načina priprave vzorcev proteinov z razgradnjo na peptide za proteomsko analizo z masnim spektrometrom: SDS PAGE in stopenjska priprava v raztopini, ki vključuje denaturacijo, redukcijo, alkilacijo proteinov in njihovo cepitev na peptide s tripsinom. Naš namen je bil skrajšati, poenostaviti in poceniti v znanstveni literaturi opisani protokol stopenjske priprave vzorcev proteinov v raztopini, vendar kljub temu ohraniti dobro razgradnjo proteinov in identifikacijo z masnim spektrometrom. V delu je tako podrobneje prikazana analiza optimizacije priprave vzorcev v raztopini: različni reagenti, različne koncentracije, združevanje korakov, dodajanje korakov, različne temperature in različno dolge inkubacije. Rezultati kažejo, da protokola ne moremo skrajšati na račun časa inkubacije tripsina, vendar pa lahko to storimo z združitvijo korakov denaturacije, redukcije in alkilacije ter skrajšanim časom inkubacije teh korakov. Kot boljši alkilacijski reagent se je v primerjavi z jodoacetamidom (IAM) zaradi manjšega števila neželenih modifikacij izkazal kloroacetamid (CAM), za oba reagenta pa je primerna koncentracija pod 10 mM. Ugotovili smo, da dodaten korak prekinitve alkilacije z ditiotreitolom (DTT) zaradi prihranka časa in kemikalij ni primeren, saj ne zmanjša števila neželenih modifikacij. Povzeti protokol smo nekoliko pocenili z zamenjavo alkilacijskih reagentov, saj je kloroacetamid (CAM) cenejši in ga je na voljo za več eksperimentov, sicer pa ceno celotnega eksperimenta določa predvsem poraba tripsina, čigar cena znaša okrog 2,23 EUR za 1 μL.

Keywords

proteomika;SDS PAGE;in-solution-digestion;reagenti;masna spektroskopija;priprava vzorcev;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2023
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[E. Zajšek]
UDC:	547.964(043.2)
COBISS:	148487171
Views:	126
Downloads:	7
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Optimization of blood plasma protein preparation by degradation to peptides in proteomic analysis solution by mass spectrometry
Secondary abstract:	In this work, we present the most common methods of protein sample preparation by degradation to peptides for proteomic analysis with a mass spectrometer: SDS PAGE and in solution digestion, which includes denaturation, reduction, alkylation of proteins and their cleavage into peptides with trypsin. Our goal was to shorten, simplify and make cheaper the protocol for stepwise preparation of protein samples in solution described in the scientific literature, but still ensure good protein degradation and identification with the mass spectrometer. The work shows in detail the analysis of the optimization of the sample preparation in solution: different reagents, different concentrations, combined steps, additional steps, different temperatures and different incubation times. The results show that the protocol cannot be shortened at the expense of the trypsin incubation time, but can be shortened by combining the denaturation, reduction, and alkylation steps, as well as by shortening the incubation time. Compared with iodoacetamide (IAM), chloroacetamide (CAM) proved to be a better alkylation reagent due to the lower number of unwanted modifications, and a concentration below 10 mM is suitable for both reagents. We found that the additional step of stopping alkylation with dithiothreitol (DTT) to save time and chemicals was not useful because it did not reduce the number of unwanted modifications. By replacing the alkylation reagents, the summary protocol became slightly cheaper because (chloroacetamide (CAM) is cheaper and more easily available for multiple experiments, otherwise the price of the whole experiment is mainly determined by the consumption of trypsin, of which price is about 2,3 EUR for 1 μL.
Secondary keywords:	proteomics;SDS PAGE;in-solution-digestion;reagents;mass spectroscopy;sample preparation;
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	1 spletni vir (1 datoteka PDF (XII, 65 f.))
ID:	18205831