Discovery of Ovarian Cancer Biomarkers in Serum Using NanoLC Electrospray Ionization TOF and FT-ICR Mass Spectrometry

Treatment of cancer patients is greatly facilitated by detection of the cancer prior to metastasis. One of the obstacles to early cancer detection is the lack of availability of biomarkers with sufficient specificity. With modern differential proteomic techniques, the potential exists to identify high specificity cancer biomarkers. We have delineated a set of protocols for the isolation and identification of serum biomarkers for ovarian cancer that exist in the low molecular weight serum fraction. After isolation of the low molecular weight fraction by ultrafiltration, the potential biomarkers are separated by reversed phase nano liquid chromatography. Detection via TOF or FT-ICR yields a data set for each sample. We compared stage III/IV ovarian cancer serum with postmenopausal age-matched controls. Using bioinformatics tools developed at Mayo, we normalized each sample for intensity and chromatographic alignment. Normalized data sets are subsequently compared and potential biomarkers identified. Several candidate biomarkers were found. One of these contains the sequence of fibrinopeptide-A known to be elevated in many types of cancer including ovarian cancer. The protocols utilized will be examined and would be applicable to a wide variety of cancers or disease states.

Download Full-text

Analysis of Albumin-Associated Peptides and Proteins from Ovarian Cancer Patients

Clinical Chemistry ◽

10.1373/clinchem.2005.052944 ◽

2005 ◽

Vol 51 (10) ◽

pp. 1933-1945 ◽

Cited By ~ 146

Author(s):

Mark S Lowenthal ◽

Arpita I Mehta ◽

Kristina Frogale ◽

Russell W Bandle ◽

Robyn P Araujo ◽

...

Keyword(s):

Ovarian Cancer ◽

Molecular Weight ◽

Western Blotting ◽

Cancer Susceptibility ◽

Solid Phase ◽

Reversed Phase ◽

Low Molecular Weight ◽

Affinity Capture ◽

Proteins And Peptides

Abstract Background: Albumin binds low–molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low–molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n= 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method. Methods: Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition. Results: In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin. Conclusion: Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.

Download Full-text

Simultaneous Determination of Human Serum Albumin and Low-Molecular-Weight Thiols after Derivatization with Monobromobimane

Molecules ◽

10.3390/molecules26113321 ◽

2021 ◽

Vol 26 (11) ◽

pp. 3321

Author(s):

Katarzyna Kurpet ◽

Rafał Głowacki ◽

Grażyna Chwatko

Keyword(s):

Molecular Weight ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Simultaneous Determination ◽

Reversed Phase ◽

Fluorescence Labeling ◽

Detector Response ◽

Low Molecular Weight

Biothiols are extremely powerful antioxidants that protect cells against the effects of oxidative stress. They are also considered relevant disease biomarkers, specifically risk factors for cardiovascular disease. In this paper, a new procedure for the simultaneous determination of human serum albumin and low-molecular-weight thiols in plasma is described. The method is based on the pre-column derivatization of analytes with a thiol-specific fluorescence labeling reagent, monobromobimane, followed by separation and quantification through reversed-phase high-performance liquid chromatography with fluorescence detection (excitation, 378 nm; emission, 492 nm). Prior to the derivatization step, the oxidized thiols are converted to their reduced forms by reductive cleavage with sodium borohydride. Linearity in the detector response for total thiols was observed in the following ranges: 1.76–30.0 mg mL−1 for human serum albumin, 0.29–5.0 nmol mL−1 for α-lipoic acid, 1.16–35 nmol mL−1 for glutathione, 9.83–450.0 nmol mL−1 for cysteine, 0.55–40.0 nmol mL−1 for homocysteine, 0.34–50.0 nmol mL−1 for N-acetyl-L-cysteine, and 1.45–45.0 nmol mL−1 for cysteinylglycine. Recovery values of 85.16–119.48% were recorded for all the analytes. The developed method is sensitive, repeatable, and linear within the expected ranges of total thiols. The devised procedure can be applied to plasma samples to monitor biochemical processes in various pathophysiological states.

Download Full-text

Proteomic Determination of Low-Molecular-Weight Glutenin Subunit Composition in Aroona Near-Isogenic Lines and Standard Wheat Cultivars

International Journal of Molecular Sciences ◽

10.3390/ijms22147709 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7709

Author(s):

Kyoungwon Cho ◽

You-Ran Jang ◽

Sun-Hyung Lim ◽

Susan B. Altenbach ◽

Yong Q. Gu ◽

...

Keyword(s):

Molecular Weight ◽

Allelic Variation ◽

Reversed Phase ◽

Glutenin Subunit ◽

Low Molecular Weight ◽

Near Isogenic Lines ◽

Wheat Cultivars ◽

Reliable Determination ◽

Isogenic Lines

The low-molecular weight glutenin subunit (LMW-GS) composition of wheat (Triticum aestivum) flour has important effects on end-use quality. However, assessing the contributions of each LMW-GS to flour quality remains challenging because of the complex LMW-GS composition and allelic variation among wheat cultivars. Therefore, accurate and reliable determination of LMW-GS alleles in germplasm remains an important challenge for wheat breeding. In this study, we used an optimized reversed-phase HPLC method and proteomics approach comprising 2-D gels coupled with liquid chromatography–tandem mass spectrometry (MS/MS) to discriminate individual LMW-GSs corresponding to alleles encoded by the Glu-A3, Glu-B3, and Glu-D3 loci in the ‘Aroona’ cultivar and 12 ‘Aroona’ near-isogenic lines (ARILs), which contain unique LMW-GS alleles in the same genetic background. The LMW-GS separation patterns for ‘Aroona’ and ARILs on chromatograms and 2-D gels were consistent with those from a set of 10 standard wheat cultivars for Glu-3. Furthermore, 12 previously uncharacterized spots in ‘Aroona’ and ARILs were excised from 2-D gels, digested with chymotrypsin, and subjected to MS/MS. We identified their gene haplotypes and created a 2-D gel map of LMW-GS alleles in the germplasm for breeding and screening for desirable LMW-GS alleles for wheat quality improvement.

Download Full-text

Validation and Application of a Derivatization-Free RP-HPLC-DAD Method for the Determination of Low Molecular Weight Salivary Metabolites

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17176158 ◽

2020 ◽

Vol 17 (17) ◽

pp. 6158

Author(s):

Beatrice Campanella ◽

Tommaso Lomonaco ◽

Edoardo Benedetti ◽

Massimo Onor ◽

Riccardo Nieri ◽

...

Keyword(s):

Molecular Weight ◽

Low Cost ◽

Reversed Phase ◽

Low Molecular Weight ◽

Single Individual ◽

Sampling Device ◽

Non Invasive ◽

Minimal Sample ◽

Whole Saliva

Saliva is an interesting, non-conventional, valuable diagnostic fluid. It can be collected using standardized sampling device; thus, its sampling is easy and non-invasive, it contains a variety of organic metabolites that reflect blood composition. The aim of this study was to validate a user-friendly method for the simultaneous determination of low molecular weight metabolites in saliva. We have optimized and validated a high throughput, direct, low-cost reversed phase liquid chromatographic method with diode array detection method without any pre- or post-column derivatization. We indexed salivary biomolecules in 35 whole non-stimulated saliva samples collected in 8 individuals in different days, including organic acids and amino acids and other carbonyl compounds. Among these, 16 whole saliva samples were collected by a single individual over three weeks before, during and after treatment with antibiotic in order to investigate the dynamics of metabolites. The concentrations of the metabolites were compared with the literature data. The multianalyte method here proposed requires a minimal sample handling and it is cost-effectiveness as it makes possible to analyze a high number of samples with basic instrumentation. The identification and quantitation of salivary metabolites may allow the definition of potential biomarkers for non-invasive “personal monitoring” during drug treatments, work out, or life habits over time.

Download Full-text

Separation of low molecular weight rapeseed proteins by RP-HPLC-DAD – a short report

Czech Journal of Food Sciences ◽

10.17221/3292-cjfs ◽

2011 ◽

Vol 24 (No. 1) ◽

pp. 41-44 ◽

Cited By ~ 1

Author(s):

A. Kosińska ◽

ChavanUD ◽

R. Amarowicz

Keyword(s):

Molecular Weight ◽

Sinapic Acid ◽

Reversed Phase ◽

Low Molecular Weight ◽

Short Report ◽

Uv Spectrum ◽

Phenolic Constituents ◽

Separation Of Proteins ◽

Two Peaks ◽

Low Molecular Weight Proteins

Low molecular weight proteins were extracted and isolated from rapeseed and analysed using the HPLC-DAD method. The separation of proteins and phenolic compounds was done on the reversed phase C<sub>18</sub> column with a gradient of acetonitrile in water. The chromatogram was characterised by two peaks of low molecular weight proteins with the retention times of 19.92 and 23.24 min. Additional three main peaks of phenolic constituents were recorded on the chromatogram. One of them with maximum of UV spectrum at 328 nm was identified as sinapic acid derivatives.

Download Full-text

Ovarian Cancer Biomarkers: Current Options and Future Promise

Journal of the National Comprehensive Cancer Network ◽

10.6004/jnccn.2008.0059 ◽

2008 ◽

Vol 6 (8) ◽

pp. 795-802 ◽

Cited By ~ 27

Author(s):

Christine M. Coticchia ◽

Jiang Yang ◽

Marsha A. Moses

Keyword(s):

Ovarian Cancer ◽

Disease Progression ◽

Cancer Detection ◽

Therapeutic Efficacy ◽

Cancer Biomarkers ◽

Cancer Diagnostics ◽

Early Disease ◽

Molecular Approaches ◽

Ovarian Cancer Diagnostics ◽

Potential Biomarkers

As more effective, less toxic cancer drugs reach patients, the need for accurate and reliable cancer diagnostics and prognostics has become widely appreciated. Nowhere is this need more dire than in ovarian cancer; here most women are diagnosed late in disease progression. The ability to sensitively and specifically predict the presence of early disease and its status, stage, and associated therapeutic efficacy has the potential to revolutionize ovarian cancer detection and treatment. This article reviews current ovarian cancer diagnostics and prognostics and potential biomarkers that are being studied and validated. Some of the most recent molecular approaches being used to identify genes and proteins are presented, which may represent the next generation of ovarian cancer diagnostics and prognostics.

Download Full-text