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Källsten, M., Pijnappel, M., Hartmann, R., Lehmann, F., Kovac, L., Lind, S. & Bergquist, J. (2019). Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates. Analytical and Bioanalytical Chemistry, 411(12), 2569-2576
Open this publication in new window or tab >>Application of triple quadrupole mass spectrometry for the characterization of antibody-drug conjugates
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2019 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 411, no 12, p. 2569-2576Article in journal (Refereed) Published
Abstract [en]

Antibody-drug conjugates (ADCs) are an inherently heterogeneous class of biotherapeutics, the development of which requires extensive characterization throughout. During the earliest phases of preclinical development, when synthetic routes towards the desired conjugate are being assessed, the main interest lies in the determination of the average drug-to-antibody ratio (DAR) of a given batch as well as information about different conjugation species. There has been a trend in mass spectrometry (MS)-based characterization of ADCs towards the use of high-resolving mass spectrometry for many of these analyses. Considering the high cost for such an instrument, the evaluation of cheaper and more accessible alternatives is highly motivated. We have therefore tested the applicability of a quadrupole mass analyzer for the aforementioned characterizations. Eight ADCs consisting of trastuzumab and varying stoichiometries of Mc-Val-Cit-PABC-monomethyl auristatin E conjugated to native cysteines were synthesized and served as test analytes. The average DAR value and molecular weights (Mw) of all detected chains from the quadrupole mass analyzer showed surprisingly high agreement with results obtained from a time-of-flight (TOF) mass analyzer and hydrophobic interaction chromatography (HIC)-derived values for all investigated ADC batches. Acquired Mw were within 80ppm of TOF-derived values, and DAR was on average within 0.32 DAR units of HIC-derived values. Quadrupole mass spectrometers therefore represent a viable alternative for the characterization of ADC in early-stage development.

Place, publisher, year, edition, pages
SPRINGER HEIDELBERG, 2019
Keywords
Reversed-phase liquid chromatography, Antibody-drug conjugates, Drug-to-antibody ratio, Triple quadrupole mass spectrometry
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-383155 (URN)10.1007/s00216-019-01699-0 (DOI)000464948100011 ()30848315 (PubMedID)
Funder
Swedish Foundation for Strategic Research , ID14-0081Swedish Foundation for Strategic Research , SB16-0039Swedish Research Council, 2015-4870
Available from: 2019-05-10 Created: 2019-05-10 Last updated: 2019-05-10Bibliographically approved
Hawkes, J. A., Sjöberg, P. J. R., Bergquist, J. & Tranvik, L. (2019). Complexity of dissolved organic matter in the molecular size dimension: insights from coupled size exclusion chromatography electrospray ionisation mass spectrometry. Faraday discussions (Online)
Open this publication in new window or tab >>Complexity of dissolved organic matter in the molecular size dimension: insights from coupled size exclusion chromatography electrospray ionisation mass spectrometry
2019 (English)In: Faraday discussions (Online), ISSN 1359-6640, E-ISSN 1364-5498Article in journal (Refereed) Epub ahead of print
Abstract [en]

This paper investigates the relationship between apparent size distribution and molecular complexity of dissolved organic matter from the natural environment. We used a high pressure size exclusion chromatography (HPSEC) method coupled to UV-Vis diode array detection (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) in order to compare the apparent size of natural organic matter, determined by HPSEC-UV and the molecular mass determined online by ESI-MS. We found that there was a clear discrepancy between the two methods, and found evidence for an important pool of organic matter that has a strong UV absorbance and no ESI-MS signal. Contrary to some previous research, we found no evidence that apparently high molecular weight organic matter is constituted by aggregates of low molecular weight (<1000 Da) material. Furthermore, our results suggest that the majority of apparent size variability within the ESI ionisable pool of organic matter is due to secondary interaction and exclusion effects on the HPSEC column, and not true differences in hydrodynamic size or intermolecular aggregation.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-384038 (URN)10.1039/c8fd00222c (DOI)
Available from: 2019-06-04 Created: 2019-06-04 Last updated: 2019-06-10Bibliographically approved
Karunasekera, H., Pettersson, J., Mi, J., Bergquist, J. & Daniel, G. (2019). Copper tolerance of the soft-rot fungus Phialophora malorum grown in-vitro revealed by microscopy and global protein expression. International Biodeterioration & Biodegradation, 137, 147-152
Open this publication in new window or tab >>Copper tolerance of the soft-rot fungus Phialophora malorum grown in-vitro revealed by microscopy and global protein expression
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2019 (English)In: International Biodeterioration & Biodegradation, ISSN 0964-8305, E-ISSN 1879-0208, Vol. 137, p. 147-152Article in journal (Refereed) Published
Abstract [en]

In this study, we used proteomics in conjunction with microscopy to study differences in the proteome and hyphal morphology of the copper tolerant soft rot fungus Phialophora malorum grown in media containing 0.064, 0.64% Cu as CuSO4. Unique proteins were found in the control and the copper-treated (0.064% CuSO4) samples. Of five unique proteins found in the 0.064% CuSO4 treated cultures, ATP synthase subunit alpha is considered to play an important role in copper tolerance as it is involved in the biosynthesis of fatty acids and steroids and may relate to morphological changes associated with hyphal cell walls of the fungus when grown in the presence of copper. ICP-AES analyses showed total mycelial Cu to increase with media Cu with 5246- and 16535 mu g Cu/g dry wt mycelia respectively found in 0.064 and 0.64% Cu-cultures after 6 weeks growth. Rubeanic acid staining of 0.064% mycelia showed Cu bound in intracellular bodies while most Cu was found as extracellular precipitates on the surfaces of hyphae in 0.64% Cu. SEM showed hyphal surfaces enrobed in fibrillar polysaccharides to which Cu was bound.

Keywords
Copper tolerance, ICP-AES, LC-MS/MS, Phialophora malorum, Proteomics, Soft rot fungi
National Category
Wood Science
Identifiers
urn:nbn:se:uu:diva-378196 (URN)10.1016/j.ibiod.2018.12.001 (DOI)000458470800017 ()
Funder
Swedish Research Council Formas, 2011-416Swedish Research Council Formas, 2011-6383-19675Swedish Research Council Formas, 621-2015-4870
Available from: 2019-03-07 Created: 2019-03-07 Last updated: 2019-03-07Bibliographically approved
Zhu, Y., Qi, X., Yu, C., Yu, S., Zhang, C., Zhang, Y., . . . Mi, J. (2019). Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB). Clinical Proteomics, 16, Article ID 12.
Open this publication in new window or tab >>Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB)
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2019 (English)In: Clinical Proteomics, ISSN 1542-6416, E-ISSN 1559-0275, Vol. 16, article id 12Article in journal (Refereed) Published
Abstract [en]

Background: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice.

Methods: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry.

Results: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC.

Conclusion: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

Place, publisher, year, edition, pages
BMC, 2019
Keywords
Esophageal squamous cell carcinoma (ESCC), Label-free quantitative proteomics, Prothymosin alpha (PTMA), Quantitative Dot Blot (QDB)
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-382384 (URN)10.1186/s12014-019-9232-6 (DOI)000463784200001 ()30988666 (PubMedID)
Available from: 2019-04-26 Created: 2019-04-26 Last updated: 2019-04-26Bibliographically approved
Valdimarsdottir, R., Valgeirsdóttir, H., Wikström, A.-K., Kallak, T. K., Elenis, E., Axelsson, O., . . . Sundström Poromaa, I. (2019). Pregnancy and neonatal complications in women with polycystic ovary syndrome in relation to second-trimester anti-Müllerian hormone levels. Reproductive Biomedicine Online, 39(1), 141-148
Open this publication in new window or tab >>Pregnancy and neonatal complications in women with polycystic ovary syndrome in relation to second-trimester anti-Müllerian hormone levels
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2019 (English)In: Reproductive Biomedicine Online, ISSN 1472-6483, E-ISSN 1472-6491, Vol. 39, no 1, p. 141-148Article in journal (Refereed) Published
Abstract [en]

RESEARCH QUESTION: An association has been found between high anti-Müllerian hormone (AMH) levels during pregnancy and the development of polycystic ovary syndrome (PCOS)-like phenotypic traits in mouse offspring. The aim of this study was to determine whether AMH levels are associated with maternal testosterone levels, and whether high AMH concentration influences the risk of developing PCOS-related adverse pregnancy outcomes.

DESIGN: Maternal serum AMH, testosterone and sex hormone binding globulin levels were measured in blood samples taken in early second-trimester pregnancies from women with PCOS (n = 159) and healthy controls matched for body mass index (n = 320). Possible associations with preeclampsia, gestational hypertension, gestational diabetes, preterm birth and birthweight was explored by logistic and linear regression models.

RESULTS: Women with PCOS had higher AMH, higher total testosterone levels and higher free androgen index than controls (P < 0.001 for all three parameters). Among women with PCOS, high testosterone levels (B = 2.7; β = 0.26; P = 0.001) and low first trimester body mass index (B = -0.5; β = -0.17; P = 0.043) remained independently associated with AMH. High AMH levels were associated with decreased risk of gestational hypertension (adjusted OR 0.55; 95% CI 0.34 to 0.87), but no association was found with other adverse pregnancy outcomes or birthweight.

CONCLUSIONS: Women with PCOS had higher AMH levels during pregnancy compared with controls, but high AMH was not associated with increased risk of adverse pregnancy outcomes or birthweight.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Birthweight, Polycystic ovary syndrome, anti-Müllerian hormone, Pregnancy complications, Testosterone
National Category
Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:uu:diva-388700 (URN)10.1016/j.rbmo.2019.02.004 (DOI)000472220300015 ()31036431 (PubMedID)
Available from: 2019-07-03 Created: 2019-07-03 Last updated: 2019-08-07Bibliographically approved
Ubhayasekera, K., Acharya, S. R. & Bergquist, J. (2018). A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites. The Analyst, 143(15), 3661-3669
Open this publication in new window or tab >>A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites
2018 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 143, no 15, p. 3661-3669Article in journal (Refereed) Published
Abstract [en]

The development of a rapid, sensitive and reliable method for the quantification of bioactive arachidonic acid metabolites (AA-metabolites) in biological samples is quite challenging due to the minute concentration, short half-life and their structural complexity arising from different isomers. In this study, a simple, fast and environmentally friendly supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method was developed and validated for simultaneous measurement of five (PGD(2), PGE(2), PGF(2), 6KetoPGF(1) and LTB4) AA-metabolites in biological samples. These analytes were extracted by protein precipitation followed by separation and quantification. The analysis was completed within 3 minutes. The matrix matched linear calibration ranged from 0.5-100 ng mL(-1) (r(2) 0.995), whilst, the limit of quantification of PGD(2), PGE(2), PGF(2), and LTB4 was 0.5 ng mL(-1) and was 2.5 ng mL(-1) for 6KetoPGF(1). The interday and intraday precisions of the method were less than 15% while the accuracy of most of the analytes varied between 83 and 109%. Finally, as a proof of concept, the method was successfully applied for the determination of eicosanoids in human samples, which expands the possibility to explore physiological states, disease phenotypes, and novel biomarkers.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2018
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-361490 (URN)10.1039/c8an00788h (DOI)000439586400019 ()29971278 (PubMedID)
Funder
Swedish Research Council, 2015-4870
Available from: 2018-09-27 Created: 2018-09-27 Last updated: 2018-09-27Bibliographically approved
de Kock, N., Acharya, S. R., Ubhayasekera, S. J. & Bergquist, J. (2018). A Novel Targeted Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid Chromatography Hyphenated to Tandem Mass Spectrometry. Scientific Reports, 8, Article ID 16993.
Open this publication in new window or tab >>A Novel Targeted Analysis of Peripheral Steroids by Ultra-Performance Supercritical Fluid Chromatography Hyphenated to Tandem Mass Spectrometry
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 16993Article in journal (Refereed) Published
Abstract [en]

Ultra-performance supercritical fluid chromatography-tandem mass spectrometry (UPSFC-MS/MS) is an alternative method for steroid analysis. Continuous development of analytical methodologies for steroid profiling is of major importance in the clinical environment to provide useful and more comprehensive data. The aim of this study was to identify and quantify a large number of endogenous steroids from the four major classes (estrogens, androgens, progestogens and corticosteroids) simultaneously within a short analytical time. This novel UPSFC-MS/MS method with electrospray in positive ionisation (ESI+) mode is robust, selective and present sufficiently high sensitivity to profile nineteen steroids in 50 mu L human plasma. Under optimised conditions, nineteen different steroids were separated with high efficiency in the multiple reaction monitoring (MRM) mode. The linearity of the method was good with correlation coefficients (R-2) in the range of 0.9983-0.9999 and with calibration range from 0.05-500 ng/mL in human plasma. The intraday and interday precision of the method, as RSD, was less than 15%. The accuracy of the nineteen analytes varied between 80 to 116%. Finally, the novel method was successfully applied for the determination of nineteen steroids within 5 minutes providing the possibility to use it for research as well as routine healthcare practice.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-371531 (URN)10.1038/s41598-018-35007-0 (DOI)000450411700024 ()30451874 (PubMedID)
Funder
Swedish Research Council, 621-2015-4870
Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-07Bibliographically approved
Cedernaes, J., Schonke, M., Westholm, J. O., Mi, J., Chibalin, A., Voisin, S., . . . Benedict, C. (2018). Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans. Science Advances, 4(8), Article ID eaar8590.
Open this publication in new window or tab >>Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans
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2018 (English)In: Science Advances, E-ISSN 2375-2548, Vol. 4, no 8, article id eaar8590Article in journal (Refereed) Published
Abstract [en]

Curtailed sleep promotes weight gain and loss of lean mass in humans, although the underlying molecular mechanisms are poorly understood. We investigated the genomic and physiological impact of acute sleep loss in peripheral tissues by obtaining adipose tissue and skeletal muscle after one night of sleep loss and after one full night of sleep. We find that acute sleep loss alters genome-wide DNA methylation in adipose tissue, and unbiased transcriptome-, protein-, and metabolite-level analyses also reveal highly tissue-specific changes that are partially reflected by altered metabolite levels in blood. We observe transcriptomic signatures of inflammation in both tissues following acute sleep loss, but changes involving the circadian clock are evident only in skeletal muscle, and we uncover molecular signatures suggestive of muscle breakdown that contrast with an anabolic adipose tissue signature. Our findings provide insight into how disruption of sleep and circadian rhythms may promote weight gain and sarcopenia.

Place, publisher, year, edition, pages
AMER ASSOC ADVANCEMENT SCIENCE, 2018
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-364473 (URN)10.1126/sciadv.aar8590 (DOI)000443498100025 ()30140739 (PubMedID)
Funder
Swedish Research Council, 2015-03100Knut and Alice Wallenberg FoundationSwedish Research Council, 2014-6888Swedish Research Council, 2016-01088Swedish Research Council, 2016-02195Swedish Research Council, 2015-4870Carl Tryggers foundation Erik, Karin och Gösta Selanders FoundationFredrik och Ingrid Thurings StiftelseLars Hierta Memorial FoundationMagnus Bergvall FoundationNovo NordiskTore Nilsons Stiftelse för medicinsk forskningSwedish Society of Medicine, SLS-694111The Swedish Brain FoundationÅke Wiberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-10-31 Created: 2018-10-31 Last updated: 2019-03-15Bibliographically approved
Bergquist, J. & Turner, C. (2018). Analytical chemistry for a sustainable society - trends and implications. Analytical and Bioanalytical Chemistry, 410(14), 3235-3237
Open this publication in new window or tab >>Analytical chemistry for a sustainable society - trends and implications
2018 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 410, no 14, p. 3235-3237Article in journal, Editorial material (Other academic) Published
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-357652 (URN)10.1007/s00216-018-1036-4 (DOI)000431401000004 ()29663055 (PubMedID)
Available from: 2018-08-28 Created: 2018-08-28 Last updated: 2018-08-28Bibliographically approved
Neupane, R., Källsten, M., Lehmann, F. & Bergquist, J. (2018). Effect of mobile phase composition on the analysis of aggregates of antibody drug conjugates (ADCs) using size exclusion chromatography. Analytical Methods, 10(9), 938-941
Open this publication in new window or tab >>Effect of mobile phase composition on the analysis of aggregates of antibody drug conjugates (ADCs) using size exclusion chromatography
2018 (English)In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 10, no 9, p. 938-941Article in journal (Refereed) Published
Abstract [en]

In this study, the effect of mobile phase composition for size exclusion chromatography (SEC) on antibody drug conjugate (ADC) aggregate analysis was investigated. The use of organic components as well as high ionic strength during aggregate analysis was demonstrated to be advantageous.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2018
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-350281 (URN)10.1039/c7ay02696j (DOI)000426696100001 ()
Funder
Swedish Research Council, SRC 2015-4870Swedish Foundation for Strategic Research , ID14-0081
Available from: 2018-05-09 Created: 2018-05-09 Last updated: 2018-05-09Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4597-041x

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