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Undin, T., Dahlin, A., Hörnaeus, K., Bergquist, J. & Bergström Lind, S. (2016). Mechanistic investigation of the on surface enzymatic digestion (oSED) protein adsorption detection method using targeted mass spectrometry. The Analyst, 141(5), 1714-1720
Open this publication in new window or tab >>Mechanistic investigation of the on surface enzymatic digestion (oSED) protein adsorption detection method using targeted mass spectrometry
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2016 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 141, no 5, p. 1714-1720Article in journal (Refereed) Published
Abstract [en]

This study describes our efforts to study some of the mechanistic aspects of the earlier established onsurface enzymatic digestion (oSED) method. In a multitude of application areas, it has become important to be able to fully characterize and understand selective protein adsorption to biomaterial surfaces for various applications, including biomedicine (implants), nanotechnology (microchip surfaces and sensors) and materials sciences. Herein, the investigation of the mechanistic aspects was based on microdialysis catheter tubes that were flushed with controlled protein solutions mimicking the extracellular fluid of the brain. The protein adsorption properties were monitored using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) with a targeted method. The temporally resolved results show that most proteins stay adsorbed onto the surface during the entire digestion process and are only cut away piece by piece, whereas smaller proteins and peptides seem to desorb rather easily from the surface. This information will simplify the interpretation of data generated using the oSED method and can also be used for the characterization of the physicochemical properties controlling the adsorption of individual proteins to specific surfaces.

National Category
Analytical Chemistry Engineering and Technology
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-248629 (URN)10.1039/c5an02091c (DOI)000371229600018 ()
Funder
Magnus Bergvall FoundationBerzelii Centre EXSELENTSwedish Research Council, 621-2011-4423
Available from: 2015-04-05 Created: 2015-04-05 Last updated: 2017-12-04Bibliographically approved
Undin, T. (2015). Characterization of Molecular Adsorption Using Liquid Chromatography and Mass Spectrometry. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Characterization of Molecular Adsorption Using Liquid Chromatography and Mass Spectrometry
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Molecular adsorption is a key feature in several disciplines of chemistry, covering as diverse fields as chromatographic separation to biomaterial development. This thesis aims at developing methods and techniques for the characterization of molecular adsorption at the liquid-solid interface. Two different experimental models were used, small molecular interaction characterization using liquid chromatography and complex protein adsorption on polymeric materials possible for biological sampling. Holistic approaches, where both detailed molecular interactions and identifications of trends, could improve the fundamental understanding of adsorption systems, were invariably part of the scientific process.

The characterization of small molecular interactions on liquid chromatography stationary phases via adsorption isotherm determination used combined data from physical phase parameters i.e. carbon loading, linear-, and nonlinear-characterization methods. These experiments were conducted on high performance liquid chromatography systems, using both ordinary reversed phase stationary phases, and hybrid phases. The expansion of the improved elusion by characteristic point (ECP) for adsorption isotherm determination, led to that previous impossible isotherm types, having inflexion points, now could be determined by the method. It also reduced errors in isotherm parameters due to the elimination of inaccurate determined retention times where the mobile phase concentration was zero.

The characterization of protein adsorption where performed in an unbiased way. Adsorbed proteins on different surfaces were identified using mass spectrometry (MS) and data dependent acquisition or a targeted method. Prior MS, an improved on surface enzymatic digestion (oSED) method was used to enable identification and quantitation of adsorbed protein originating from ventricular cerebrospinal fluid (vCSF). oSED was found to be able to experimentally determine large variations in protein adsorption characteristics between native and coated polycarbonate surfaces in contact with vCSF. The method was also confirmed being mechanistic in favor of enzymatic digestion of the proteins adsorbed on a surface, rather than a prior desorption into solution before digestion.

An improvement of the overall understanding of adsorption systems was not only achieved with the oSED method as a promising tool for characterization of protein adsorption on arbitrary surfaces, but also the use of linear and nonlinear approaches in stationary phase characterization that strengthened drawn conclusions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. p. 50
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1248
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-249003 (URN)978-91-554-9235-9 (ISBN)
Public defence
2015-05-22, A1:111a, Husargatan 3, BMC, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2015-04-27 Created: 2015-04-09 Last updated: 2015-07-07
Chu, J., Undin, T., Lind, S., Dahlin, A. & Hjort, K. (2015). Influence of different pluronic surface modifications and pressure on microdialysis protein extraction efficiency. Biomedical microdevices (Print)
Open this publication in new window or tab >>Influence of different pluronic surface modifications and pressure on microdialysis protein extraction efficiency
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2015 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781Article in journal (Refereed) Submitted
Abstract [en]

There is growing interest in using microdialysis (MD) for monitoring larger and more complexmolecules such as neuropeptides and proteins. This promotes the use of MD membranes withmolecular weight cut off (MWCO) of 100 kDa. Hence, the hydrodynamic property of themembrane goes to ultrafiltration, making the sampling more sensitive to pressure changes. Also,despite the large membrane pore size, studies have shown that membrane biofouling still leads tounstable catheter performance. Our objective is to study in vitro how four kinds of surfacemodifications (Pluronic L31, L44, F87 and F127+L31) affect the fluid recovery (FR) andextraction efficiency (EE) of 100 kDa MWCO MD catheters, under controlled pressure. Apressure chamber was employed to facilitate the tests, using as MD sample a protein standardwith proteins of similar concentrations as in human cerebral spinal fluid. The collected dialysatefractions were examined for FR and EE. Targeted mass spectrometry analysed the EE ofindividual proteins and peptides. The thicker the pluronic adsorption layer, the less thehydrodynamic diameter of the membrane pores, leading to lower and more stable FR. The foursurface modifications had three different behaviours: Pluronic F127 + L31 showed similarbehavior to the Pluronic F127 and the native original membrane; Pluronic F87 showed acontinuous EE increase with pressure; Pluronic L31 and L44 showed similar EE values, whichwere stable with pressure. Different surface modifications are clearly selective to differentproteins and peptides. We conclude that a pluronic surface modification could provide MDsampling with more stable FR, and more stable or enhanced EE with high FR, depending on theobjective of the sampling.

Place, publisher, year, edition, pages
Springer: , 2015
Keyword
microdialysis, surface modification, poloxamer, protein, extraction efficiency
National Category
Nano Technology
Research subject
Engineering Science with specialization in Microsystems Technology; Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-261557 (URN)
Funder
Berzelii Centre EXSELENT
Available from: 2015-09-01 Created: 2015-09-01 Last updated: 2017-12-04Bibliographically approved
Chu, J., Undin, T., Bergström Lind, S., Hjort, K. & Dahlin, A. (2015). Influence of surface modification and static pressure on microdialysis protein extraction efficiency. Biomedical microdevices (Print), 17(5), Article ID UNSP 96.
Open this publication in new window or tab >>Influence of surface modification and static pressure on microdialysis protein extraction efficiency
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2015 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 17, no 5, article id UNSP 96Article in journal (Refereed) Published
Abstract [en]

There is growing interest in using microdialysis (MD) for monitoring larger and more complexmolecules such as neuropeptides and proteins. This promotes the use of MD membranes withmolecular weight cut off (MWCO) of 100 kDa or above. The hydrodynamic property of themembrane goes to ultrafiltration or beyond, making the MD catheters more sensitive to pressure.In the meantime, despite the large pore size, studies have shown that membrane biofouling stilllead to unstable catheter performance. The objective is to study in vitro how 500 kDa dextranand Poloxamer 407 surface modification affect the fluid recovery (FR) and extraction efficiency(EE) of 100 kDa MWCO MD catheters. A pressure chamber was designed to facilitate the tests,using as MD sample a protein standard with similar concentrations as in human cerebral spinalfluid, comparing native and Poloxamer 407 modified MD catheters. The collected dialysatefractions were examined for FR and protein EE, employing Dot-it Spot-it Protein Assay for totalprotein EE and targeted mass spectrometry (MS) for EE of individual proteins and peptides. TheFR results suggested that the surface modified catheters were less sensitive to the pressure andprovide higher precision, and provided a FR closer to 100%. The surface modification did notshow a significant effect on the protein EE. The average total protein EE of surface modifiedcatheters was slightly higher than that of the native ones. The MS EE data of individual proteinsshowed a clear trend of complex response in EE with pressure.

Place, publisher, year, edition, pages
Springer: , 2015
Keyword
microdialysis, surface modification, poloxamer, protein, extraction efficiency
National Category
Nano Technology
Research subject
Engineering Science with specialization in Microsystems Technology
Identifiers
urn:nbn:se:uu:diva-261031 (URN)10.1007/s10544-015-0005-3 (DOI)000362281200013 ()26342494 (PubMedID)
Funder
Berzelii Centre EXSELENTSwedish Research Council, P29797-1Åke Wiberg FoundationMagnus Bergvall FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2015-08-28 Created: 2015-08-28 Last updated: 2017-12-04Bibliographically approved
Undin, T., Bergström Lind, S. & Dahlin, A. P. (2015). MS for investigation of time-dependent protein adsorption on surfaces in complex biological samples. Future Science OA, Article ID 32.
Open this publication in new window or tab >>MS for investigation of time-dependent protein adsorption on surfaces in complex biological samples
2015 (English)In: Future Science OA, ISSN 2056-5623, article id 32Article in journal (Refereed) Published
Abstract [en]

Aim: This study aims at developing a nondestructive way for investigating proteinadsorption on surfaces such as biomaterials using mass spectrometry. Methods: Ventricular cerebrospinal fluid in contact with poly carbonate membranes were usedas adsorption templates and on-surface enzymatic digestion was applied to desorbproteins and cleave them into peptides. Mass spectrometric analysis provided bothprotein identification and determination of protein specific adsorption behavior. Results: In general, the adsorption increased with incubation time but also proteinspecifictime-resolved adsorption patterns from the complex protein solutionwere discovered. Conclusion: The method developed is a promising tool for thecharacterization of biofouling, which sometimes causes rejection and encapsulationof implants and can be used as complement to other surface analytical techniques.

One problem associated with artificial materials in the body is that proteins in thebody interact with the surface, which sometimes causes rejection of the implant.In this study, a method for investigating the time-dependent protein adsorptionon a surface originating from a complex biological protein solution was developed.Compared with other surface analyses, this method can identify what proteins thatadsorbs on the surface. In addition, determination of protein-specific adsorptionbehavior in relation to incubation was possible

National Category
Chemical Sciences Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-262022 (URN)10.4155/fso.15.32 (DOI)
Funder
Åke Wiberg Foundation, M14–0127Magnus Bergvall Foundation, 2014-00367
Available from: 2015-09-07 Created: 2015-09-07 Last updated: 2017-01-24Bibliographically approved
Chu, J., Undin, T., Dahlin, A., Wang, C., Park, J. & Hjort, K. (2015). Protein Desalination Chip for Mass Spectrometry Sample Preparation. In: : . Paper presented at The 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015. October 25-29, 2015. Gyeongju, Korea.. MicroTAS
Open this publication in new window or tab >>Protein Desalination Chip for Mass Spectrometry Sample Preparation
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2015 (English)Conference paper, Oral presentation with published abstract (Refereed)
Abstract [en]

This work focuses on desalination of a protein sample in a lab-on-chip device using the ion concentration polarization (ICP) technique. It was demonstrated with a salt containing buffer with four proteins and two peptides of concentrations typical to cerebrospinal fluid (CSF). Not only was the output desalinated but its protein concentration with large molecular weight (MW) was as much as 3 times higher for the largest protein compared to the original. We conclude that ICP based microfluidic chips have great potential for desalination and protein concentration in microdialysis sampling coupled to mass spectroscopy (MS).

Place, publisher, year, edition, pages
MicroTAS: , 2015
Keyword
Ion concentration polarization, Desalination, Protein, Mass spectroscopy
National Category
Nano Technology
Research subject
Engineering Science with specialization in Microsystems Technology; Engineering Science with specialization in Materials Science
Identifiers
urn:nbn:se:uu:diva-261067 (URN)
Conference
The 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2015. October 25-29, 2015. Gyeongju, Korea.
Available from: 2015-08-28 Created: 2015-08-28 Last updated: 2016-01-05
Undin, T., Samuelsson, J., Törncrona, A. & Fornstedt, T. (2013). Evaluation of a combined linear–nonlinear approach for column characterization using modern alkaline-stable columns as model. Journal of Separation Science, 36(11), 1753-1761
Open this publication in new window or tab >>Evaluation of a combined linear–nonlinear approach for column characterization using modern alkaline-stable columns as model
2013 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 36, no 11, p. 1753-1761Article in journal (Refereed) Published
Abstract [en]

This study investigates if deeper understanding is achieved when combining nonlinear and linear chromatographic column characterization methods. As test systems, two hybrid columns (Phenomenex Gemini-NX C18 and Kromasil Eternity C18) and one classic one (Kromasil-C18) were selected. The nonlinear methods were based on firm adsorption theory and involved determination of adsorption isotherms followed by calculations with a new numerical tool, adsorption energy distribution, on probe components at different pH values. The linear methods involved the hydrophobic subtraction model and selected probe components retention factors as a function of pH. The combined analysis indicated that both complementary and confirmative information can be achieved regarding the actual model systems.

Keyword
Adsorption isotherms, Characterization, Linear methods, Nonlinear methods, Retention
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-202052 (URN)10.1002/jssc.201201132 (DOI)000319919900009 ()
Available from: 2013-06-19 Created: 2013-06-19 Last updated: 2017-12-06Bibliographically approved
Undin, T., Dahlin, A., Bergquist, J. & Wetterhall, M. (2013). Investigation of Time Dependent Competitive Protein Adsorption to Surfaces Using Mass Spectrometry. In: : . Paper presented at HUPO.
Open this publication in new window or tab >>Investigation of Time Dependent Competitive Protein Adsorption to Surfaces Using Mass Spectrometry
2013 (English)Conference paper, Oral presentation with published abstract (Refereed)
Abstract [en]

Introduction

Surfaces in a complex protein solution will adsorb proteins. This event is fast and dynamic and triggers a biological response against the inserted/implanted biomaterial that eventually will lead to biofouling and encapsulation. This affects the properties of the inserted devices, such as hampered membrane functions of microdialysis (MD) probes or distortion in response of biosensors.

Methods

Untreated and coated filtration membranes were used as adsorption templates for human ventricular cerebrospinal fluid (vCSF). After adsorption in an incubation chamber, the membranes were washed, dried and the proteins were reduced, alkylated and digested. The sample preparation procedure was conducted according to an on-surface enzymatic digestion (oSED) protocol previously described by our group. The oSED digests were analyzed by nanoLC ESI-MS/MS using a 7T hybrid LTQ FT and Velos pro orbitrap mass spectrometer.

Preliminary Data

In this study, we present a time resolved map of protein adsorption. Non-coated and tri-block polymer coated, polycarbonate membranes was used as templates. As expected, a time and surface property dependent protein adsorption relationship was observed. It is not surprising that the degree of protein binding onto modified and non-modified surfaces was dependent on the properties of the protein as well as the properties of the surface. The process of biofouling for in vivo inserted materials can be postponed and thereby increasing the lifetime and use of e.g. microdialysis probes for patient monitoring. The preliminary data are very promising making it possible to identify a spectra of adsorbed proteins on different surfaces in a time dependent way

Keyword
Competitive, Surface, Adsorption
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-214392 (URN)
Conference
HUPO
Funder
Berzelii Centre EXSELENT
Available from: 2014-01-08 Created: 2014-01-08 Last updated: 2014-01-09Bibliographically approved
Undin, T., Bergquist, J., Dahlin, A. P. & Wetterhall, M. (2012). Competitive Protein Adsorption as Observed and Quantified by - Surface Enzymatic Digestion (oSED) and Mass Spectrometry. In: 60th ASMS Conference on Mass Spectrometry and Allied Topics, May 20 - 24, Vancouver, Canada. Paper presented at 60th ASMS Conference on Mass Spectrometry and Allied Topics, May 20 - 24, Vancouver, Canada.
Open this publication in new window or tab >>Competitive Protein Adsorption as Observed and Quantified by - Surface Enzymatic Digestion (oSED) and Mass Spectrometry
2012 (English)In: 60th ASMS Conference on Mass Spectrometry and Allied Topics, May 20 - 24, Vancouver, Canada, 2012Conference paper, Published paper (Refereed)
National Category
Analytical Chemistry Engineering and Technology
Research subject
Engineering Science with specialization in Microsystems Technology
Identifiers
urn:nbn:se:uu:diva-188861 (URN)
Conference
60th ASMS Conference on Mass Spectrometry and Allied Topics, May 20 - 24, Vancouver, Canada
Available from: 2012-12-20 Created: 2012-12-20 Last updated: 2013-03-04Bibliographically approved
Enmark, M., Samuelsson, J., Undin, T. & Fornstedt, T. (2011). Characterization of an unusual adsorption behavior of racemic methyl-mandelate on a tris-(3,5-dimethylphenyl) carbamoyl cellulose chiral stationary phase. Journal of Chromatography A, 1218(38), 6688-6696
Open this publication in new window or tab >>Characterization of an unusual adsorption behavior of racemic methyl-mandelate on a tris-(3,5-dimethylphenyl) carbamoyl cellulose chiral stationary phase
2011 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1218, no 38, p. 6688-6696Article in journal (Refereed) Published
Abstract [en]

An interesting adsorption behavior of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase was theoretically and experimentally investigated. The overloaded band of the more retained enantiomer had a peculiar shape indicating a type V adsorption isotherm whereas the overloaded band of the less retained enantiomer had a normal shape indicating a type I adsorption behavior. For a closer characterization of this separation, adsorption isotherms were determined and analyzed using an approach were Scatchard plots and adsorption energy distribution (AED) calculations are combined for a deeper analysis. It was found that the less retained enantiomer was best described by a Tóth adsorption isotherm while the second one was best described with a bi-Moreau adsorption isotherm. The latter model comprises non-ideal adsorbate–adsorbate interactions, providing an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated using synthetically generated raw adsorption slope data. It was found that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and found to be exothermic; i.e. the adsorbate–adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process revealed that the average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.

Keyword
Chiral separation, Perturbation peak method, Adsorption energy distribution, Stochastic modeling, Tóth adsorption model, Moreau adsorption model
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-161205 (URN)10.1016/j.chroma.2011.07.064 (DOI)
Available from: 2011-11-09 Created: 2011-11-09 Last updated: 2018-01-23Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3861-9338

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