Liquid Chromatography-selected Reaction Monitoring Mass Research Articles

B vitamin quantification in lentil seed tissues using ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry

Lentils are a nutritious food in the human diet. High in protein and with low glycemic index, lentils are also a source of folate and other B vitamins. Understanding variability in B vitamin contents among lentils will allow breeders to select for increased levels. We analyzed 34 cultivated and three wild genotypes for vitamins B1, B2, B3, B5, B6, B7, and B9 in the cotyledons and seed coats. Variation for all B vitamins was observed across the genotypes. Cotyledons had higher concentrations of B1 and B3, while seed coats had higher concentrations of B2, B5, B6, and B9. Wild accessions had the highest concentrations of vitamin B9 and were among the highest for B2. Differential distribution of B vitamins across seed tissues and lentil genotypes has implications for consumption and for breeding. There is useful genetic variability which could be used to increase B vitamin levels in future lentil varieties.

Comparative analysis of heat‐stress‐induced abscisic acid and heat shock protein responses among pea varieties

AbstractHeat shock proteins (HSPs) and abscisic acid (ABA) play important roles in plant heat responses but have not been extensively studied in pea (Pisum sativum L.), whose heat susceptibility is well known. In this study, four pea varieties varying in heat tolerance based on field trials were evaluated. Plants were heat stressed for 3, 6, 12, or 24 h at 38 °C before pollination. Anther and stipule RNA from the same flowering node were sampled for transcriptional profiling of PsHSP18.1 and PsHSP71.2. Additional stipules were sampled for the quantification of ABA concentration and its five key catabolites from the four major ABA catabolic pathways by ultra‐high performance liquid chromatography–selected reaction monitoring mass spectrometry (UHPLC‐SRM/MS). The transcription of both HSP genes was upregulated because of heat stress (HS). In stipules, the upregulation was greatest at 3 h HS, whereas in anthers, the induced transcription was similar among different hours of HS. Likewise, more ABA accumulated in the ABA metabolism pool because of HS, and the ABA response started rapidly after 3 h of treatment. Heat‐tolerant varieties had a higher ABA synthesis and turnover rate at 3 h HS than their respective heat‐susceptible counterparts. This study provides new insights into different heat tolerance among Canadian pea varieties regarding HSP and ABA hormone regulation.

Validated B vitamin quantification from lentils by selected reaction monitoring mass spectrometry

A validated method for B vitamin separation and quantification from lentil seeds using ultra high performance liquid chromatography-selected reaction monitoring mass spectrometry (UHPLC-SRM MS) was reported. The use of three enzymes (acid phosphatase, β-glucosidase, and rat serum) with a 4 h incubation was sufficient to convert bound B vitamins into their free forms. Twenty B vitamers were selected and a 5-min UHPLC-SRM MS method was optimized for rapid analysis. This method was applied to quantify B vitamin concentration during lentil seed germination over a 5-day period. Total B vitamins increased up to 1.5-fold on day 5 (from 39.2 µg/g to 60.6 µg/g of dry weight) comparing with dry seeds. Vitamin B5 (pantothenic acid) was the most abundant B vitamin in both dry seeds (34.2%) and in germinated seeds (17.7%-24.5% of total B vitamins); B8 (biotin) and B12 (cyanocobalamin) were not detected in lentil samples.

Targeted quantification of B vitamins using ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry in faba bean seeds

Ultra-performance liquid chromatography-selected reaction monitoring mass spectrometry (UPLC-SRM MS) was used for targeted quantification of water-soluble B vitamins from seeds of 30 genotypes of faba bean with contrasting anti-nutritional profiles. Sixteen vitamers from seven B vitamins were quantified from whole seeds, seed coats and cotyledons. A single-step extraction approach with the use of four enzymes (acid phosphatase, β-glucosidase, protease and rat serum) and 2 h incubation at 37 °C was used. The total B vitamin concentrations ranged from 23.08 to 39.26 μg/g in whole seeds, 11.51 to 30.31 μg/g in seed coats and 23.56 to 32.66 μg/g in cotyledons. Significant genotype by environment interaction effects were observed for all B vitamins. Our results indicated low-tannin genotypes had high B5 content, whereas tannin-containing genotypes had high B1, B2, B6, and B9 content, particularly in seed coats. The analysis of a large number of faba bean germplasm here suggests that its seeds are a rich source of vitamins B1, B2, B3 and B5. The increased consumption of faba bean seeds and ingredients in the human diet can provide enhanced dietary B vitamins.

Quantification of Vitamin B12-Related Proteins in Marine Microbial Systems Using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry-based proteomic approaches to studying microbial systems enable assessment of taxonomically resolved functional capacity. A subset of these proteomic approaches are absolutely quantitative, enabling comparisons of protein expression patterns between different studies and across environments. This chapter outlines a method for applying quantitative assays in marine microbial communities, using proteins involved in vitamin B12 (cobalamin) utilization and production as specific examples. This approach involves identifying important protein targets, determining taxonomic resolution of the required assays, identifying suitable peptides, developing and optimizing liquid chromatography-selected reaction monitoring mass spectrometry assays (LC-SRM-MS), and processing the resulting data. Implementing the method outlined here results in measurements (fmol diagnostic peptide per μg of total bulk protein) that, in this case, define the nutritional status of microbial community members with respect to vitamin B12, and are comparable across and between marine microbial systems.

Quantitative proteomics of breast tumors: Tissue quality assessment to clinical biomarkers.

Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) is not only a proven tool for clinical chemistry, but also a versatile method to enhance the capability to quantify biomarkers for tumor biology research. As the treatment of cancer continues to evolve, the ability to assess multiple biomarkers to assign cancer phenotypes based on the genetic background and the signaling of the individual tumor becomes paramount to our ability to treat the patient. In breast cancer, the American Society of Clinical Oncology has defined biomarkers for patient assessment to guide selection of therapy: estrogen receptor, progesterone receptor, and the HER2/Neu receptor tyrosine kinase; therefore, these proteins were selected for LC-SRM assay development. Detailed molecular characterization of these proteins is necessary for patient treatment, so expression and phosphorylation assays have been developed and applied. In addition, other LC-SRM assays were developed to further evaluate tumor biology (e.g. Ki-67 for proliferation and vimentin for tumor aggressiveness related to the epithelial-to-mesenchymal transition). These measurements combined with biomarkers for tissue quality and histological content are implemented in a three-tier multiplexed assay platform, which is translated from cell line models into frozen tumor tissues banked from breast cancer patients.

Development of Phage-Based Antibody Fragment Reagents for Affinity Enrichment of Bacterial Immunoglobulin G Binding Proteins.

Disease and death caused by bacterial infections are global health problems. Effective bacterial strategies are required to promote survival and proliferation within a human host, and it is important to explore how this adaption occurs. However, the detection and quantification of bacterial virulence factors in complex biological samples are technically demanding challenges. These can be addressed by combining targeted affinity enrichment of antibodies with the sensitivity of liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS). However, many virulence factors have evolved properties that make specific detection by conventional antibodies difficult. We here present an antibody format that is particularly well suited for detection and analysis of immunoglobulin G (IgG)-binding virulence factors. As proof of concept, we have generated single chain fragment variable (scFv) antibodies that specifically target the IgG-binding surface proteins M1 and H of Streptococcus pyogenes. The binding ability of the developed scFv is demonstrated against both recombinant soluble protein M1 and H as well as the intact surface proteins on a wild-type S. pyogenes strain. Additionally, the capacity of the developed scFv antibodies to enrich their target proteins from both simple and complex backgrounds, thereby allowing for detection and quantification with LC-SRM MS, was demonstrated. We have established a workflow that allows for affinity enrichment of bacterial virulence factors.

Plasma QconCATs reveal a gender-specific proteomic signature in apheresis platelet plasma supernatants.

Gender differences in the proteome composition of apheresis platelet supernatants have always been postulated, and might underlie a higher risk of adverse reactions when transfusing apheresis products from female donors. Preliminary proteomic studies provided an overview of gender-dependent relative compositional differences in the proteome of apheresis platelet supernatants during routine storage in the blood bank. Here we apply a proteomics approach for absolute quantitation of approximately 100 proteins in apheresis platelet supernatants from male and female donors at storage days 1 and 5. Absolute quantitative proteomic analyses allowed us to confirm and expand on previous observations about gender and storage-dependency of platelet supernatant protein profiles.

A simple and robust UPLC-SRM/MS method to quantify urinary eicosanoids

Eicosanoids are key mediators and regulators of inflammation and oxidative stress often used as biomarkers for diseases and pathological conditions such as cardiovascular and pulmonary diseases and cancer. Analytically, comprehensive and robust quantification of different eicosanoid species in a multi-method approach is problematic because most of these compounds are relatively unstable and may differ in their chemical properties. Here we describe a novel ultra-performance liquid chromatography-selected reaction monitoring mass spectroscopy (UPLC-SRM/MS) method for simultaneous quantification of key urinary eicosanoids, including the prostaglandins (PG) tetranor PGE-M, 8-iso-, and 2,3-dinor-8-iso-PGF(2α); the thromboxanes (TXs) 11-dehydro- and 2,3-dinor-TXB₂; leukotriene E₄; and 12-hydroxyeicosatetraenoic acid. In contrast to previous methods, which used time-consuming and complex solid phase extraction, we prepared samples with a simple liquid/liquid extraction procedure. Because collision-induced dissociation produced characteristic product ions for all analytes, no derivatization step for SRM/MS analysis was necessary. Analytes were separated with a short UPLC reversed-phase column (1.7 µm particles), allowing shorter run times than conventional HPLC columns. The method was validated and applied to human urine samples showing excellent precision, accuracy, detection limits, and robustness. In summary, the developed method allows robust and sensitive profiling of urinary eicosanoid species, making it a useful and valuable tool for biomarker profiling in clinical/toxicological studies.

Measurement of Fractional Synthetic Rates of Multiple Protein Analytes by Triple Quadrupole Mass Spectrometry

Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS. Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject. The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation. The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.

Development of a Chip/Chip/SRM Platform Using Digital Chip Isoelectric Focusing and LC-Chip Mass Spectrometry for Enrichment and Quantitation of Low Abundance Protein Biomarkers in Human Plasma

Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ∼1-2.5 ng/mL with a CV of ∼13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.

Detection of diarrhetic shellfish poisoning toxins from tropical shellfish using liquid chromatography-selected reaction monitoring mass spectrometry.

A negative mode liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) method was developed to detect low concentrations of the diarrhetic shellfish poisoning (DSP) toxins okadaic acid and dinophysistoxin-1 (DTX-1). Detection relies upon monitoring the transition of negative precursor ions [M - H]- to a common fragment ion of m/z 255. Our limit of detection for okadaic acid with this method is 0.5 pg on column. LC-SRM MS has allowed us to detect persistent, low concentrations of DSP toxins from Singapore shellfish.

Persistent low concentrations of diarrhetic shellfish toxins in green mussels Perna viridis from the Johor Strait, Singapore:first record of diarrhetic shellfish toxins from South-East Asia

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 181:257-268 (1999) - doi:10.3354/meps181257 Persistent low concentrations of diarrhetic shellfish toxins in green mussels Perna viridis from the Johor Strait, Singapore: first record of diarrhetic shellfish toxins from South-East Asia Michael James Holmes1,*, Serena Lay Ming Teo1, Fu Chin Lee1, Hong Woo Khoo2 1Bioscience Centre and 2Department of Biological Sciences, National University of Singapore, 119260 Singapore *E-mail: bschmj@nus.edu.sg ABSTRACT: Liquid chromatography-selected reaction monitoring mass spectrometry was used to identify and quantify Diarrhetic Shellfish Poisoning (DSP) toxins for the first time from tropical shellfish. Persistent, low concentrations of okadaic acid, 6 isomers of okadaic acid, and 5 isomers of dinophysistoxin-1 (DTX-1) were detected from green mussels Perna viridis from 3 sites in the Johor Strait, Singapore, between October 1995 and December 1997. Isomers of okadaic acid and DTX-1 generally occurred in higher concentrations than okadaic acid. The highest concentration of any single DSP toxin detected from Singapore shellfish was 97 ng g-1 mussel digestive tissue (wet weight) of an isomer of DTX-1 (DTX-1a). The maximum concentration of okadaic acid detected was 24 ng g-1 digestive tissue. These concentrations are well below the generally recommended limit for consumption of DSP toxins for humans (~1 µg toxin g-1 digestive tissue). Naturally contaminated mussels rapidly depurated okadaic acid when held in a laboratory aquarium; however, okadaic acid and some isomers of okadaic acid and DTX-1 could still be detected (<1 ng g-1) after 32 d. Phytoplankton samples from the Johor Strait were examined for the origin of the DSP toxins found in green mussels. Four species of dinophysoid dinoflagellates were found, with Dinophysis caudata the most frequent and abundant species; although, cell densities did not exceed 5 cells l-1. Five hundred D. caudata were micropipetted from phytoplankton samples and analysed for DSP toxins. Okadaic acid was detected (7 x 10-14 g cell-1) from D. caudata but no isomers of okadaic acid, DTX-1 or isomers of DTX-1. D. caudata were found in the gut contents of Singapore green mussels and probably contribute to the contamination of these shellfish; however, other as yet unidentified sources of DSP toxins may exist in the Johor Strait. KEY WORDS: Diarrhetic Shellfish Poisoning · Okadaic acid · Dinophysistoxin · Perna viridis · Dinophysis caudata · Dinoflagellate · Mass spectrometry Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 181. Publication date: May 18, 1999 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1999 Inter-Research.