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Mass Spectrometry-based characterization of the vitreous phosphoproteome
Ist Super Sanit, Dept Hematol Oncol & Mol Med, Rome, Italy.
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2010 (English)In: Proteomics Clinical Applications, ISSN 1862-8346, Vol. 4, no 10-11, 839-846 p.Article in journal (Refereed) Published
Abstract [en]

Purpose: The vitreous gel is a highly hydrated extracellular matrix containing many proteins. These proteins are likely accumulated in the vitreous by local secretion, filtration from the blood, or diffusion from the surrounding tissues and vasculature, and may be altered in disease state. In the last several years, several reports of large-scale profiling of vitreous proteins have been published; however, there is little information on the characterization of the phosphoproteome of vitreous. Here, we sought to identify phosphopeptides and their phosphorylation sites from vitreous.

Experimental design: We used titanium dioxide (TiO2) to enrich phosphopeptides from vitreous and identified them by LC-MS/MS.

Results: We identified 85 unique phosphopeptides and the phosphorylation sites from 44 proteins.

Conclusions and clinical relevance: We present a method for characterization of phosphoproteome from vitreous samples using current MS technologies and yielded an initial assessment of the phosphoprotein/peptide content of human vitreous, thus providing important biological information toward further understanding of the post-translational modifications of vitreous proteins and their functional significance in disease.

Place, publisher, year, edition, pages
2010. Vol. 4, no 10-11, 839-846 p.
Keyword [en]
Crystallin; LTQ-Orbitrap; Phosphopeptide; Titanium dioxide; Vitreous
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:su:diva-56867DOI: 10.1002/prca.201000032OAI: oai:DiVA.org:su-56867DiVA: diva2:413511
Available from: 2011-04-28 Created: 2011-04-28 Last updated: 2011-05-03Bibliographically approved
In thesis
1. Colloidal/Solid Phase Extraction (C/S PE) Methods Based on Hydrogel Nanoparticles, Titanium dioxide microparticles and Empore Membranes Applied to Biological and Environmental Matrices
Open this publication in new window or tab >>Colloidal/Solid Phase Extraction (C/S PE) Methods Based on Hydrogel Nanoparticles, Titanium dioxide microparticles and Empore Membranes Applied to Biological and Environmental Matrices
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of the work described in this thesis was to create and develop novel technologies in order to overcome barriers and hurdles that analytical chemistry faces focusing on sample extraction. In paper I dye containing amine groups (e.g. Acid Black 48, Remazol Brilliant Blue R) were coupled to NIPA/Acrylic acid (AAc) particles by condensation of the amine group and the carboxylic group. The high affinity between dyes and proteins allow for fast kinetics and complete depletion of the supernatant and protection of the captured analyte from enzymatic degradation. The ability of particles to capture and concentrate analytes was tested against a panel of low abundance, labile tumor relevant biomarkers and in serum. Results indicate that the nanoparticles increased the sensitivity limit of mass spectrometry analysis and that the dye based baits have extremely high affinity for the target analytes so that particles capture all the analyte present in solution. Biomarker harvesting nanoparticles may be useful for discovery of novel diagnostic analytes, can increase the sensitivity of detection for analytical methods such as immunoassays and MS, and protect labile biomarkers from degradation during collection, shipment and storage. In paper II and paper III, applications of hydrogel nanoparticles to serum samples from cancer patients are reported. Hydrogel nanoparticles were integrated in a mass spectrometry based workflow for the discovery of candidate biomarkers. Lists of candidate biomarkers were identified that are under verification and validation. In paper IV and V hydrogel nanoparticles functionalized with dyes, were employed to increase the sensitivity of diagnostic test for Lyme disease and to detect human growth hormone (hGH) in urine samples. In paper VI, titanium dioxide (TiO2) microparticles were used to pack fused silica capillary column and used to capture and enrich phosphopeptides in vitreous samples. In paper VII, Empore disk membranes were used to capture organophosphates (OPEs) flame retardant from air samples. Empore disk membranes were used as on- line extraction followed by reverse phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS) analysis. Optimized “geometry” settings were used to strip semi volatile and volatile compounds from C8 membrane. This novel design allowed for a better analyte focusing in the HPLC column, reduced the volume of the organic solvent employed for the extraction and the analysis time, and eliminated sample contamination, and loss of analyte.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholms University, 2011. 110 p.
Keyword
biomarkers, nanogels, solid-phase extraction, organophosphate esters, peptides, mass spectrometry
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56873 (URN)978-91-7447-231-8 (ISBN)
Public defence
2011-06-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
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Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 5: Manuscript. Paper 6: Manuscript.Available from: 2011-05-10 Created: 2011-04-28 Last updated: 2011-05-02Bibliographically approved

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