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Methods for protein analysis by capillary electrophoresis and mass spectrometry
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Applied Physical Chemistry. (Åsa Emmer, analytisk kemi)ORCID iD: 0000-0003-3091-3882
2018 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Protein analysis is important to understanding biological systems, but sample diversity necessitates a multitude of analysis techniques and methods. Challenges that are addressed include analysis of low abundance samples, fractionation to reduce sample complexity, and automation to reduce time and cost.

Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important technique for protein characterization. In Paper I, the sensitivity of MALDI-MS was enhanced through the fabrication of a hydrophobic coating for the MALDI target plate, yielding analyte concentration. The plate outperformed a commercial concentration plate.

Capillary electrophoresis (CE) separation offers low sample consumption and high efficiency, and in Paper II, offline CE-MALDI-MS fractionation was employed. A robot system for automation was constructed and used in analysis of spermatophore proteins from the butterfly Pieris napi. The robot was also used in automated on-target trypsin digestion under a lid of liquid fluorocarbons, a simpler and cheaper alternative to controlled humidity chambers. An indication of indigenous proteolysis of the sample was seen.

Electrospray ionization (ESI) is the other technique for protein analysis in MS. In Paper III, the biomarker protein osteopontin (OPN) was analyzed by ESI-MS in order to find suitable conditions for its detection. A preliminary optimization of solvents and ionization conditions was done, and tandem MS (MSn) performed to increase the reliability of identification.

Abstract [sv]

Proteinanalys är viktigt för att förstå biologiska system, men mångfalden av prov kräver en mängd olika analystekniker och metoder. Utmaningar som tas upp inkluderar analys av små provmängder, fraktionering för att minska provkomplexiteten, och automatisering för att minska tidsåtgång och kostnad.

Matris-assisterad laserjoniserings-masspektrometri (MALDI-MS) är en viktig teknik för proteinkarakterisering. I Artikel I förbättrades känsligheten i MALDI-MS genom tillverkning av en hydrofob beläggning på MALDI-provplattan, vilket gav en koncentration av provet. Provplattan gav bättre resultat än en kommersiell koncentrationsprovplatta.

Kapillärelektroforesseparation (CE) har låg provåtgång och hög separationseffektivitet och i Artikel II användes offline CE-MALDI-MS-fraktionering. Ett robotsystem för automatisering konstruerades och användes för analys av spermatoforproteiner från fjärilen Pieris napi. Roboten användes även i automatiserad trypsinklyvning under en yta av en flytande fluorkolförening, ett billigare alternativ tilli nkubationskammare med kontrollerad luftfuktighet. En indikation på naturlig enzymatisk proteinklyvning i provet hittades.

Elektrospray jonisering (ESI) är den andra tekniken för proteinanalys i MS. I Artikel III analyserades biomarkören osteopontin (OPN) med ESI-MS för att hitta lämpliga förhållanden för dess detektion. En preliminär optimering av lösningsmedel och jonisationsförhållanden gjordes, och tandem-MS (MSn) utfördes för att öka identifikationens tillförlitlighet.

Place, publisher, year, edition, pages
US-AB , 2018. , p. 34
Series
TRITA-CBH-FOU ; 2018-60TRITA-CBH-FOUTRITA-CBH-FOUTRITA-CBH-FOUTRITA-CBH-FOUTRITA-CBH-FOU
Series
TRITA-CBH-FOU ; 2018-60
Keywords [en]
MALDI; Concentration plates; Alkyl ketene dimer; CE-MALDI-MS2; On-target digestion; Automation; Robot; Pieris napi; spermatophore; ESI-MS2; Osteopontin
Keywords [sv]
MALDI; Koncentrationsprovplatta; alkylketenedimer; CE-MALDI-MS2; enzymklyvning på provplatta; Automatisering; Robot; Pieris napi; Spermatofor; ESI-MS2; Osteopontin
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-239311ISBN: 978-91-7873-039-1 (print)OAI: oai:DiVA.org:kth-239311DiVA, id: diva2:1264470
Presentation
2018-12-19, K53, Teknikringen 28, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

Full text will not be uploaded due to unpublished results. QC 20181121

Available from: 2018-11-21 Created: 2018-11-20 Last updated: 2018-11-21Bibliographically approved
List of papers
1. Simple and environmentally friendly fabrication of superhydrophobic alkyl ketene dimer coated MALDI concentration plates
Open this publication in new window or tab >>Simple and environmentally friendly fabrication of superhydrophobic alkyl ketene dimer coated MALDI concentration plates
2017 (English)In: Journal of the American Society for Mass Spectrometry, ISSN 1044-0305, E-ISSN 1879-1123, Vol. 28, no 8, p. 1733-1736Article in journal (Refereed) Published
Abstract [en]

Here we present a method to manufacture peptide-concentrating MALDI-plates with alkyl ketene dimer (AKD) as a new superhydrophobic coating. The fabrication of the hydrophobic plates included application of AKD by airbrush, and negative contact printing to generate the concentration sites. Deposited sample droplets were contained within the prestructured sites, and self-adjusted onto the site if slightly misplaced. No AKD contamination was observed, and the plates could easily be cleaned and regenerated. The S/N values for four model peptides was about twice as high compared with a standard steel plate and a commercial concentration plate.

Place, publisher, year, edition, pages
Springer, 2017
Keywords
MALDI Concentration plates Alkyl ketene dimer
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-204944 (URN)10.1007/s13361-017-1657-4 (DOI)000405486200025 ()2-s2.0-85024100522 (Scopus ID)
Funder
Carl Tryggers foundation , CTS 13:117 15:146
Note

QC 20170419

Available from: 2017-04-05 Created: 2017-04-05 Last updated: 2018-11-20Bibliographically approved
2. An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi
Open this publication in new window or tab >>An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi
(English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376XArticle in journal (Refereed) Accepted
Abstract [en]

A method for off-line CE‑MALDI‑TOF-MS and MS2, and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS2 spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.

Keywords
CE MALDI MS2; On target digestion; automation; robot; Pieris napi; spermatophore
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-239353 (URN)
Note

QC 20181122

Available from: 2018-11-21 Created: 2018-11-21 Last updated: 2018-11-22Bibliographically approved
3. ESI-MSn Analysis of Recombinant Human Osteopontin
Open this publication in new window or tab >>ESI-MSn Analysis of Recombinant Human Osteopontin
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The low-abundance protein osteopontin is implicated in several serious diseases, where its concentration andglycosylation patterns might be analyzed for its use as a biomarker. The glycosylation has previously been studied andcharacterized mainly on digested protein. Allowing analysis of glycosylation using the intact protein would reduce theworkload and analysis time, as well as introducing less potential sources of error and bias. Here, the detection of intactosteopontin by ESI-MS is presented. By using a matrix with a high proportion of isopropanol, osteopontin could be detectedand fragmented in tandem MS at 10 µg/mL by direct infusion. A lower osteopontin mass was also present in the sample. Theresults open the possibilities of further analysis of osteopontin by tandem MS and suggests a reporter ion.

National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-239355 (URN)
Projects
Methods for protein analysis by capillary electrophoresis and mass spectrometry
Note

QC 20181122

Available from: 2018-11-21 Created: 2018-11-21 Last updated: 2018-11-22Bibliographically approved

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The full text will be freely available from 2019-12-19 11:25
Available from 2019-12-19 11:25

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