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Digestion of Enolase and Carbonic Anhydrase as Model Proteins for Therapeutic Proteins in Blood Plasma with Immobilized Thermolysin and Quantification of Some of the Peptides by LC/LC-MS/MS
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för analytisk kemi.
2014 (Engelska)Ingår i: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 77, nr 1-2, s. 59-74Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

There is a need for fast method development in the early drug discovery phase of therapeutic proteins. Thermolysin has not been used for quantification of proteins in blood plasma earlier. It is a thermostable protease which permits the use of high temperatures for fast hydrolysis of proteins. Model proteins were digested with immobilized thermolysin on agarose gel. Protein-specific peptides were selected for quantitation and quantified based on stable isotope dilution. Protein digests of blood plasma were cleaned and separated using an automated LC/LC-MS/MS system. Essential digestion parameters that influence thermolysin hydrolytic activity were optimized for high peptide yield. The validated methods were selective, linear, precise and accurate with a limit of detection of 2 nM for both proteins. The proposed strategy for method development could be valuable for quantification of proteins in blood plasma samples. The study underscores and discusses important features of the enzymatic digestion and chromatographic separation.

Ort, förlag, år, upplaga, sidor
2014. Vol. 77, nr 1-2, s. 59-74
Nyckelord [en]
On-line two-dimensional liquid chromatography, Mass spectrometry, Hydrolysis with thermolysin, Enzyme immobilization, Quantification of proteins
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:su:diva-100656DOI: 10.1007/s10337-013-2562-zISI: 000329230500007OAI: oai:DiVA.org:su-100656DiVA, id: diva2:696037
Anmärkning

AuthorCount:4;

Tillgänglig från: 2014-02-12 Skapad: 2014-02-10 Senast uppdaterad: 2017-12-06Bibliografiskt granskad
Ingår i avhandling
1. Analysis of drugs and proteins in biological matrices, using different types of sample preparations and mass spectrometry
Öppna denna publikation i ny flik eller fönster >>Analysis of drugs and proteins in biological matrices, using different types of sample preparations and mass spectrometry
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis describes bioanalysis of small and large molecules in biological matrices and includes screening of illegal drugs in urine with high resolution mass spectrometer, bioanalysis of MTH1 inhibitors in mice plasma and quantification of proteins in plasma and cell lysates.

Screening of illegal drugs was based on high resolving power mass spectrometer (QTOF) and the results were compared to immunoassays. For the study, the nine most commonly abused drugs were selected for screening of a large number of authentic urine samples. Evaluation of the screening results showed that the QTOF generated a low rate of false positive and false negative results and can be used as an alternative or a complementary to immunoassays.

In another study, a bioanalytical method for the two new anticancer targets TH588 and TH287 was developed and validated. The compounds inhibit the MTH1 protein that is required for cancer cell survival. To study the pharmacokinetic properties of the substances, a bioanalytical method was developed using a fast sample preparation followed by LC-MS/MS analysis. Validation showed that the method was linear, accurate and precise in the studied concentration range. Stability tests showed that the substances were stable in mice plasma and under different storage conditions. The study also addresses other important factors such as solubility, chromatography and fragmentation mechanisms.

Two other studies focused on quantification of specific proteins in biological specimens; plasma and bacterial lysates. The matrices are rich in endogenous proteins making quantitative analysis of target proteins challenging. Therefore a new strategy is proposed that combines known procedures in a way it has not been used before. The methods are based on quantification with isotopically labelled peptides added after proteolytic digestion of the sample with immobilized thermolysin or pepsin. The sample digest was analysed on LC-MS/MS and LC/LC-MS/MS systems.

Ort, förlag, år, upplaga, sidor
Stockholm: Department of Analytical Chemistry, Stockholm Universtiy, 2014. s. 59
Nyckelord
Bioanalysis, drugs, proteins, liquid chromatography, two-dimensional liquid chromatography, mass spectrometry
Nationell ämneskategori
Analytisk kemi
Forskningsämne
analytisk kemi
Identifikatorer
urn:nbn:se:su:diva-107689 (URN)978-91-7447-984-3 (ISBN)
Disputation
2014-11-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 13:00 (Svenska)
Opponent
Handledare
Anmärkning

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 2: Manuscript.

Tillgänglig från: 2014-10-16 Skapad: 2014-09-24 Senast uppdaterad: 2014-10-23Bibliografiskt granskad

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