Quantification of glycopeptides by multiple reaction monitoring liquid chromatography/tandem mass spectrometry

Protein glycosylation has a major influence on functions of proteins. Studies have shown that aberrations in glycosylation are indicative of disease conditions. This has prompted major research activities for comparative studies of glycoproteins in biological samples. Multiple reaction monitoring (MRM) is a highly sensitive technique which has been recently explored for quantitative proteomics. In this work, MRM was adopted for quantification of glycopeptides derived from both model glycoproteins and depleted human blood serum using glycan oxonium ions as transitions. The utilization of oxonium ions aids in identifying the different types of glycans bound to peptide backbones. MRM experiments were optimized by evaluating different parameters that have a major influence on quantification of glycopeptides, which include MRM time segments, number of transitions, and normalized collision energies. The results indicate that oxonium ions could be adopted for the characterization and quantification of glycopeptides in general, eliminating the need to select specific transitions for individual precursor ions. Also, the specificity increased with the number of transitions and a more sensitive analysis can be obtained by providing specific time segments. This approach can be applied to comparative and quantitative studies of glycopeptides in biological samples as illustrated for the case of depleted blood serum sample. Copyright © 2012 John Wiley & Sons, Ltd.     

Response normalized liquid chromatography nanospray ionization mass spectrometry

The widely different LC-MS response observed for many structurally different compounds limits the use of LC-MS in full scan detection mode for quantitative determination of drugs and metabolites without using reference standard. The recently introduced nanospray ionization (NSI) technique shows comparable MS response for some compounds under non-LC-MS conditions. However, in the presence of numerous endogenous compounds commonly associated with biological samples such as urine, plasma, and bile, LC-MS is required to separate, detect, identify, and measure individual analytes. An LC-NSI-MS system was devised and the MS response obtained in this system for a variety of pharmaceutical drugs and their metabolites. The set-up involves two high-performance liquid chromatography (HPLC) systems, a chip-based NSI source and a quadrupole-time-of-flight (Q-TOF) mass spectrometer. Herein this is referred to as the response normalized-liquid chromatography NSI-MS (RNLC-NSI-MS) system. One HPLC unit performs the analytical separation, while the other unit adds solvent post-column with an exact reverse of the mobile phase composition such that the final composition entering the NSI source is isocratic throughout the entire HPLC run. The data obtained from four different structural classes of compounds [vicriviroc (VCV), desloratadine (DL), tolbutamide, and cocaine] and their metabolites indicate that by maintaining the solvent composition unchanged across the HPLC run, the influence of the solvent environment on the ionization efficiency is minimized. In comparison to responses obtained from radiochromatograms, responses from conventional LC-ESI-MS overestimated the VCV and DL responses, respectively, by 6- and 20-fold. Although VCV and DL responses obtained using LC-NSI-MS are within 2- to 6-fold from the respective radiochromatographic responses, the response normalization modification results in nearly uniform LC-NSI-MS response for all compounds evaluated.     

Studies of cisplatin and hemoglobin interactions using nanospray mass spectrometry and liquid chromatography with inductively-coupled plasma mass spectrometry

Interactions of cisplatin with hemoglobin (Hb) were studied using both nanoelectrospray mass spectrometry (nanoESI-MS) and a combination of size exclusion high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICPMS). Size exclusion HPLC separation of free and protein-bound cisplatin followed by simultaneous monitoring of 195Pt and 57Fe demonstrated the presence of Hb-bound Pt complexes. Nanospray quadrupole time-of-flight mass spectrometry studies of the Hb-cisplatin complexes further demonstrated the specific binding of cisplatin to the alpha-chain, heme-alpha, beta-chain, and heme-beta units of hemoglobin. Accurate mass measurements and tandem mass spectrometry information confirmed the Hb-cisplatin complexes. The formation of Hb-cisplatin complexes was observed at the sub-microM to microM concentration levels of cisplatin, which are relevant to clinical levels. These findings and the techniques developed for cisplatin-Hb interaction studies are useful for understanding of drug-protein interactions.     

Analysis of bioactive eicosanoids in equine plasma by stable isotope dilution reversed-phase liquid chromatography/multiple reaction monitoring mass spectrometry

Oxidative metabolites of arachidonic acid (AA) are implicated in inflammation. Thus, we evaluated cycloxygenases (COXs) and lipoxygenases (LOs) mediated metabolism of AA to eicosanoids in equine plasma. Eicosanoids were extracted from plasma by two liquid-liquid extraction (LLE) steps; first was by chloroform/isopropanol and second by methyl-tert-butyl ether. For identification and quantification of 25 eicosanoids, a highly specific, selective and sensitive stable isotope dilution liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometric (MS) method was developed. To avoid artifact formation of eicosanoids, deferoxamine was added to plasma to chelate residual transition metal ions. The calibration curve showed excellent linearity within 0.1 to 10 ng/mL. Slopes of the calibration curves generated by adding known quantities of eicosanoids in plasma were higher than those prepared in methanol/mobile phase A. Addition of deferoxamine decreased the slope of calibration curves generated using plasma. Limit of detection (LOD) was 1-10 pg on-column for 25 different eicosanoids. Inter-day accuracy was 86-111%, whereas intra-day accuracy was from 88-110%, and precision did not exceed 15% for all quality control (QC) samples. To evaluate the formation of eicosanoids, AA was exogenously added or endogenous AA was released from esterified lipids by calcium ionophore (CI) A23187 treatment of equine whole blood. Pre-treatment of equine whole blood with dexamethasone (DEX) significantly inhibited AA or CI A23187- mediated formation of eicosanoids. The validated method is now employed in studies undertaken to better understand the mechanism of action and pharmacokinetics/pharmacodynamics of eicosanoids after administration of glucocorticoids to horses. This method is reliably reproducible.     

Identification of phosphorylation sites in proteins by nanospray quadrupole ion trap mass spectrometry

A method is described for identifying serine phosphorylation sites in proteins, based on conventional (32)P labeling followed by electrophoretic separation, 'in-gel' digestion with a protease, peptide extraction, reversed-phase high-performance liquid chromatographic separation and collection and off-line analysis of the radioactive fractions by nanospray ion trap mass spectrometry. The method was successfully applied to the identification of three phosphorylation sites in two proteins which were subjected to in vitro phosphorylation under physiological conditions. Different combinations of the various scanning modes of the ion trap, including high-resolution, multiple subfragmentation (or MS(n)) and fast scan analysis, were employed to identify the phosphopeptides, determine their sequence and localize the exact site of phosphorylation. 'Blind' fragmentation using fast scans was used to analyze a phosphopeptide which was undetectable in other scanning modes. The sequence, phosphorylation site and double cysteine modification of the potassium adduct of a peptide containing 35 residues were also determined by multiple fragmentation. The results not only support the validity of the proposed method for routine identification of phosphorylation sites, but also demonstrate the exceptional capability of off-line ion trap mass spectrometry in combination with nanospray ionization for performing very detailed studies on the structure of peptides.     

Identification of multiple components in Guanxinning injection using hydrophilic interaction liquid chromatography/time-of-flight mass spectrometry and reversed-phase liquid chromatography/time-of-flight mass spectrometry

An approach for the identification of multiple components in traditional Chinese medicine injections (TCMIs) using a combination of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled with time-of-flight mass spectrometry (TOFMS) was developed for the quality control of Guanxinning injection (GXNI), a widely used TCMI, composed of Salvia miltiorrhiza and Ligusticum Chuanxiong. A total of 50 compounds from five compound classes, including saccharides, amino acids, organic acids, phenolic acids and phthalides, were identified or tentatively characterized on the basis of accurate mass measurements and subsequent TOFMS product ions. Six groups of isomers of phenolic acids and saccharides were tentatively distinguished. It was observed that the ESI-TOFMS fragmentation behavior of phthalides was different in negative and positive ion mode, and the fragmentation pathways were tentatively elucidated using structurally-relevant product ions. Several highly polar constituents were characterized for the first time from GXNI by HILIC/TOFMS. In addition, all the constituents identified from GXNI were further assigned in the two individual crude drugs. The integrated strategy has provided a powerful approach for the separation and identification of the multiple components in GXNI, and it has also assisted in the establishment of methods for the comprehensive safety and quality evaluation of TCMIs.     

Workplace monitoring of isocyanates using ion trap liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/ion trap tandem mass spectrometry method was developed for the qualitative and quantitative detection of isocyanates in air. The method is based on derivatization of isocyanates with 1-(2-methoxyphenyl)piperazine during air sampling. The extracts are analyzed using an ion trap LC/MS system equipped with an electrospray (ESI) ion source. The method shows high linearity, specificity, accuracy and precision. The limits of detection are 40x to 55x lower than with UV-based methods.     

CTP synthetase activity assay by liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode

Cytidine 5′‐triphosphate synthetase (CTPS) is known to be a central enzyme in the de novo synthesis of CTP. We have recently demonstrated that a deficiency in CTPS1 is associated with an impaired capacity of activated lymphocytes to proliferate leading to a combined immunodeficiency disease. In order to better document its role in immunomodulation, we developed a method for measuring CTPS activity in human lymphocytes. Using liquid chromatography‐mass spectrometry, we quantified CTPS activity by measuring CTP in cell lysates. A stable isotope analog of CTP served as internal standard. We characterized the kinetic parameters V_max and K_m of CTPS and verified that an inhibition of the enzyme activity was induced after 3‐deazauridine (3DAU) treatment, a known inhibitor of CTPS. We then determined CTPS activity in healthy volunteers, in a family whose child displayed a homozygous mutation in CTPS1 gene and in patients who had developed or not a chronic lung allograft dysfunction (CLAD) after lung transplantation. Linearity of the CTP determination was observed up to 451 μmol/L, with accuracy in the 15% tolerance range. Michaelis‐Menten kinetics for lysates of resting cells were K_m=280±310 μmol/L for UTP, V_max=83±20 pmol/min and, for lysates of activated PBMCs, K_m=230±280 μmol/L for UTP, V_max=379±90 pmol/min. Treatment by 3DAU and homozygous mutation in CTPS1 gene abolished the induction of CTPS activity associated with cell stimulation, and CTPS activity was significantly reduced in the patients who developed CLAD. We conclude that this test is suitable to reveal the involvement of CTPS alteration in immunodeficiency.     

Quantification of monosaccharides through multiple‐reaction monitoring liquid chromatography/mass spectrometry using an aminopropyl column

A simple, sensitive, and reproducible quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was designed for the simultaneous quantification of monosaccharides derived from glycoprotein and blood serum using a multiple‐reaction monitoring (MRM) approach. Sialic acids and neutral monosaccharides were efficiently separated using an amino‐bonded silica phase column. Neutral monosaccharide molecules were detected as their aldol acetate anion adducts [M + CH₃CO₂]^? using electrospray ionization in negative ion MRM mode, while sialic acids were detected as deprotonated ions [M–H]^?. The new method did not require a reduction step, and exhibited very high sensitivity to carbohydrates with limits of detection of 1 pg for the sugars studied. The linearity of the described approach spanned over three orders of magnitude (pg to ng). The method was validated for monosaccharides originating from N‐linked glycans attached to glycoproteins and glycoproteins found in human blood serum. The method effectively quantified monosaccharides originating from as little as 1 µg of glycoprotein and 5 µL of blood serum. The method was robust, reproducible, and highly sensitive. It did not require reduction, derivatization or postcolumn addition of reagents. Copyright © 2010 John Wiley & Sons, Ltd.     

Sensitive, preclinical detection of prions in brain by nanospray liquid chromatography/tandem mass spectrometry

More sensitive detection of prions in brain is important because it would allow early detection of disease in young animals and assure a safer food supply. We have quantitated the amount of proteinase K-resistant prion protein (PrP 27-30) by use of nano-scale liquid chromatography coupled to tandem mass spectrometry using the multiple reaction monitoring mode of operation. We used a method based on the detection of VVEQMCTTQYQK (residues 209-220) obtained by reduction, alkylation and digestion with trypsin. Quantitation of the amount of PrP 27-30 in the brains of Syrian hamsters was possible as early as 24 h after inoculation. Our results show sensitive detection of 180 fmol of PrP 27-30 per g brain (wet weight) as early as 24 h after inoculation. Clinical symptoms are not observed until 9 weeks after inoculation.     

Measuring long-chain acyl-coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Long-chain acyl-coenzymes A (acyl-CoAs) (LCACoA) are the activated forms of long-chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl-CoA ([U-(13) C]16-CoA) and oleoyl-CoA ([U-(13) C]18:1-CoA) using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl-CoA ([U(-13)C]16-CoA) and oleoyl-CoA ([U(-13)C]18:1-CoA) using ultra-performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14-CoA, C16-CoA, C16:1-CoA, C18-CoA, C18:1-CoA, C18:2-CoA, C20-CoA). The molecules are separated on a reversed-phase UPLC column using a binary gradient with ammonium hydroxide (NH(4) OH) in water and NH(4) OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M+2+H](+) and [U(13)C]16-CoA and [U(13)C]18:1-CoA were monitored as [M+16+H](+) and [M+18+H](+), respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U(13) C]16-CoA and [U(13) C]18:1-CoA during a low dose intravenous infusion of [U(13) C]palmitate and [U(13) C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool.     

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

A rapid and sensitive method for the quantification of ganciclovir in plasma using liquid chromatography/selected reaction monitoring/mass spectrometry

A method using reversed-phase liquid chromatography coupled with electrospray ionization and selected reaction monitoring mass spectrometry has been developed for the quantitative analysis of ganciclovir in rat plasma. Acyclovir, a structurally related analog of ganciclovir, was used as the internal standard. A small volume of plasma (50 microL) was spiked with the internal standard and plasma proteins were precipitated by methanol. The supernatant was dried under nitrogen, and then reconstituted in water. The use of liquid chromatography/selected reaction monitoring/mass spectrometry effectively eliminated potential interference from endogenous constituents in the plasma. This highly selective and sensitive method made it possible to analyze plasma ganciclovir with a lower limit of quantitation of 10 ng/mL. The assay was reproducible and linear in the range 10-10,000 ng/mL. The precision and accuracy values were in the range 2.0-6.9% and 89.0-109.6%, respectively. The analyte recovery was greater than 88%. This method was successfully used to monitor the pharmacokinetic profile of ganciclovir in normal rats following intraperitoneal administration of the drug.     

Deciphering chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma by liquid chromatography with transformed multiple reaction monitoring mass spectrometry

In this study, we propose an integrated strategy for the efficient identification and quantification of herbal constituents using liquid chromatography with mass spectrometry. First, liquid chromatography with quadrupole time‐of‐flight mass spectrometry was employed for the chemical profiling of herbs, where a targeted following nontargeted approach was developed to detect trace constituents by using structural correlations and extracted ion chromatograms. Next, ion pairs and parameters of MS² of quadrupole time‐of‐flight mass spectrometry were selected to design multiple reaction monitoring transitions for the identified compounds on liquid chromatography with triple quadrupole mass spectrometry. The relative concentration of each constituent was then calculated using a semiquantitative calibration curve. The proposed strategy was applied in a study of chemical interactions between Glycyrrhizae Radix and Coptidis Rhizoma. A total of 140 compounds were identified or tentatively characterized from the herbs, 132 of which were relatively quantified. The visualized quantitative results clearly showed codecoction produced significant constituent concentration variations especially for those with a low polarity. The case study also indicated that the present methodology could provide a reliable, accurate, and labor‐saving solution for chemical studies of herbal medicines.