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Zhou, Y., Romson, J. & Emmer, Å. (2019). An antibody-free sample pretreatment method for osteopontin combined with MALDI-TOF MS/MS analysis. PLoS ONE, 14(3), Article ID e0213405.
Open this publication in new window or tab >>An antibody-free sample pretreatment method for osteopontin combined with MALDI-TOF MS/MS analysis
2019 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 3, article id e0213405Article in journal (Refereed) Published
Abstract [en]

Osteopontin is an osteoblast-secreted protein with an aspartic acid-rich, highly phosphorylated, and glycosylated structure. Osteopontin can easily bind to integrins, tumor cells, extracellular matrix and calcium, and is related to bone diseases, various cancers, inflammation etc. Here, DEAE-Cibacron blue 3GA was used to extract recombinant osteopontin from human plasma, and to deplete abundant plasma proteins with an antibody-free method. Using selected buffer systems, osteopontin and human serum albumin could be bound to DEAE-Cibacron blue 3GA, while immunoglobulin G was excluded. The bound osteopontin could then be separated from albumin by using different sequential elution buffers. By this method, 1 μg/mL recombinant osteopontin could be separated from the major part of the most abundant proteins in human plasma. After trypsin digestion, the extracted osteopontin could be successfully detected and identified by MALDI-TOF MS/MS using the m/z 1854.898 peptide and its fragments.

Place, publisher, year, edition, pages
Public Library of Science, 2019
National Category
Analytical Chemistry
Research subject
Chemistry
Identifiers
urn:nbn:se:kth:diva-249443 (URN)10.1371/journal.pone.0213405 (DOI)000460638800082 ()30845167 (PubMedID)2-s2.0-85062607743 (Scopus ID)
Note

QC 20190522

Available from: 2019-04-12 Created: 2019-04-12 Last updated: 2019-05-22Bibliographically approved
Romson, J., Jacksen, J. & Emmer, Å. (2019). An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi. Journal of chromatography. B, 1104, 228-233
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
2019 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 1104, p. 228-233Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
CE-MALDI-MS2, On-target digestion, Automation, Robot, Pieris napi, Spermatophore
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-244140 (URN)10.1016/j.jchromb.2018.11.021 (DOI)000456890000029 ()30530115 (PubMedID)2-s2.0-85057615872 (Scopus ID)
Note

QC 20190218

Available from: 2019-02-18 Created: 2019-02-18 Last updated: 2019-02-18Bibliographically approved
Romson, J., Jacksén, J. & Emmer, Å. An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi. Journal of chromatography. B
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
Romson, J. & Emmer, Å.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
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3091-3882

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