Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis

Glycosylation is a common post-translational modification that can add complexity to the proteome of many cell types. We used enzymatic and chemical methods of deglycosylation to treat a heavily glycosylated exoproteome sample from the filamentous fungus Trichoderma reesei. Deglycosylated samples were resolved on one-dimensional (1-D) and two-dimensional (2-D) gels in order to determine the effect of deglycosylation on the electrophoresis patterns and on the ability to identify proteins by peptide mass matching using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of in-gel tryptic digests. We found that deglycosylation of the protein sample resulted in different protein patterns on 1-D and 2-D gels, reduced the complexity of gel patterns, and enhanced the protein identification of some proteins via MALDI-TOF-MS. Deglycosylation with trifluoromethanesulfonic acid (TFMS) was found to be more effective than enzymatic treatments. These deglycosylation techniques may be employed in whole proteome analysis to locate glycosylated proteins and assist in their identification by MS.     

2-DE and MALDI-TOF-MS analysis of therapeutic fusion protein abatacept

2-DE and MALDI-TOF MS are useful techniques for the quality evaluation of medicinal products derived from recombinant DNA technology. The principal objective of this study has been to evaluate the suitability of 2-DE in combination with MALDI-TOF MS for the quality study of the therapeutic recombinant protein, abatacept. 1-DE SDS-PAGE, under reducing and nonreducing conditions, and 2-DE analysis were used for the assessment of M(r) , pI, and enzymatic deglycosylation efficiency of abatacept. 2-DE allowed the assessment of product identity, purity, charge heterogeneity, isoform pattern, and post-translational modifications. Furthermore, optimization of the deglycosylation procedure, charge heterogeneity, and sample preparation for the subsequent MALDI-TOF MS analysis has been addressed. PMF analysis allowed rapid identity confirmation of abatacept.     

A potential pitfall in 18O-based N-linked glycosylation site mapping

A common procedure for identifying N-linked glycosylation sites involves tryptic digestion of the glycoprotein, followed by the conversion of glycosylated asparagine residues into (18)O-labeled aspartic acids by PNGase F digestion in (18)O water. The 3 Da mass tag created by this process is readily observable by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, and is often used to identify the sites of N-linked glycosylation. While using this procedure, we noticed that 60% of the asparagines identified as being glycosylated were not part of the consensus sequence required for N-linked glycosylation, and thus were not biologically possible. Investigation into the source of this unacceptably high false positive rate demonstrated that even after reversed-phase cleanup and heat denaturation, the trypsin used for proteolysis was still active and led to the incorporation of (18)O into the C-termini of the peptides during the deglycosylation step. The resulting mass shift accounted for most of the false positive sites, as the database search algorithm confused it with an (18)O-labeled Asp residue near the C-terminus of a peptide. This problem can be overcome by eliminating trypsin from the solution prior to performing the deglycosylation process, by resuspending the peptides in natural abundance water following deglycosylation, or by allowing (18)O incorporation into the C-terminus as a variable modification during the database search. These methods have been demonstrated on a model protein, and are applicable to the analyses of glycoproteins that are digested with trypsin or another serine protease prior to enzymatic release of the carbohydrate side chains. This study should alert investigators in the field to this potential and unexpected pitfall and provide strategies to overcome this phenomenon.     

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano- or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL^?1, respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL^?1 and the analysis of urine samples collected from horses administered rhEPO (Eprex?) or DPO (Aranesp?) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available.     

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.     

Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry

Protein citrullination originates from enzymatic deimination of polypeptide‐bound arginine and is involved in various biological processes during health and disease. However, tools required for a detailed and targeted proteomic analysis of citrullinated proteins in situ, including their citrullination sites, are limited. A widely used technique for detection of citrullinated proteins relies on antibody staining after specific derivatization of citrulline residues by 2,3‐butanedione and antipyrine. We have recently reported on the details of this reaction. Here, we show that this chemical modification can be utilized to specifically detect and identify citrullinated peptides and their citrullination sites by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Using model compounds, we demonstrate that in collision‐induced dissociation (CID) a specific, modification‐derived fragment ion appears as the dominating signal at m/z 201.1 in the MS/MS spectra. When applying electron transfer dissociation (ETD), however, the chemical modification of citrulline remained intact and extensive sequence coverage allowed identification of peptides and their citrullination sites. Therefore, LC/MS/MS analysis with alternating CID and ETD has been performed, using CID for specific, signature ion‐based detection of derivatized citrullinated peptides and ETD for sequence determination. The usefulness of this targeted analysis was demonstrated by identifying citrullination sites in myelin basic protein deiminated in vitro. Combining antibody‐based enrichment of chemically modified citrulline‐containing peptides with specific mass spectrometric detection will increase the potential of such a targeted analysis of protein citrullination in the future. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of metabolites of geniposide in rat urine using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Geniposide, an iridoid glycoside, is an important and characteristic compound in the fruits of Gardenia jasminoides Ellis, a commonly used medicinal herb in Chinese traditional and folk medicine for the treatment of inflammation and jaundice. However, few studies have been carried out on the metabolism of geniposide. In this study, we have established a rapid and sensitive method using ultra‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (UPLC/ESI‐QTOF‐MS) for analysis of the metabolic profile of geniposide in rat urine after oral administration. A total of ten metabolites were detected and identified by comparing their fragmentation patterns with that of geniposide using Metabolynx? and MassFragment? software tools. The results revealed that the principal metabolism pathways of geniposide in rat occurred after deglycosylation of the irdoid glycoside take place and this is followed by glucuronidation and the pyran‐ring cleavages. The major metabolite, the glucuronic acid conjugate of genipin as observed in vivo, was further confirmed by the in vitro enzymatic study. The results of this work have demonstrated the feasibility of the UPLC/ESI‐QTOF‐MS approach for rapid and reliable characterization of metabolites from iridoid compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation of melatonin and n‐acetylserotonin in human plasma by nanoflow LC‐MS/MS and electrospray LC‐MS/MS

Melatonin (MEL) and its chemical precursor N‐acetylserotonin (NAS) are believed to be potential biomarkers for sleep‐related disorders. Measurement of these compounds, however, has proven to be difficult due to their low circulating levels, especially that of NAS. Few methods offer the sensitivity, specificity and dynamic range needed to monitor MEL and its precursors and metabolites in small blood samples, such as those obtained from pediatric patients. In support of our ongoing study to determine the safety, tolerability and PK dosing strategies for MEL in treating insomnia in children with autism spectrum disorder, two highly sensitive LC‐MS/MS assays were developed for the quantitation of MEL and precursor NAS at pg/mL levels in small volumes of human plasma. A validated electrospray ionization (ESI) method was used to quantitate high levels of MEL in PK studies, and a validated nanospray (nESI) method was developed for quantitation of MEL and NAS at endogenous levels. In both assays, plasma samples were processed by centrifugal membrane dialysis after addition of stable isotopic internal standards, and the components were separated by either conventional LC using a Waters SymmetryShield RP18 column (2.1 × 100 mm, 3.5 µm) or on a polyimide‐coated, fused‐silica capillary self‐packed with 17 cm AquaC18 (3 µm, 125 Å). Quantitation was done using the SRM transitions m/z 233 → 174 and m/z 219 → 160 for MEL and NAS, respectively. The analytical response ratio versus concentration curves were linear for MEL (nanoflow LC: 11.7–1165 pg/mL, LC: 1165–116500 pg/mL) and for NAS (nanoflow LC: 11.0–1095 pg/mL). Copyright © 2012 John Wiley & Sons, Ltd.     

GC–MS combined with chemometric techniques for the quality control and original discrimination of Curcumae longae rhizome: Analysis of essential oils

Curcumae longae rhizome is a widely used traditional herb in many countries. Various geographical origins of this herb might lead to diversity or instability of the herbal quality. The objective of this work was to establish the chemical fingerprints for quality control and find the chemical markers for discriminating these herbs from different origins. First, chemical fingerprints of essential oil of 24 C. longae rhizome from four different geographical origins in China were determined by GC–MS. Then, pattern recognition techniques were introduced to analyze these abundant chemical data in depth; hierarchical cluster analysis was used to sort samples into groups by measuring their similarities, and principal component analysis and partial least‐squares discriminate analysis were applied to find the main chemical markers for discriminating these samples. Curcumae longae rhizome from Guangxi province had the highest essential oil yield (4.32 ± 1.45%). A total of 46 volatile compounds were identified in total. Consistent results were obtained to show that C. longae rhizome samples could be successfully grouped according to their origins, and turmerone, ar‐turmerone, and zingiberene were the characteristic components for discriminating these samples of various geographical origins and for quality control. This finding revealed that fingerprinting analysis based on GC–MS coupled with chemometric techniques could provide a reliable platform to discriminate herbs from different origins, which is a benefit for quality control.     

溶胶凝胶微流控酶反应器用于蛋白质肽谱分析的研究

The silica-based poly(dimethylsiloxane)(PDMS)microfluidic enzymatic reactor was reported along with itsanalytical features in coupling with MALDI TOF and ESI MS.Microfluidic chip was fabricated using PDMS cast-ing and O_2-plasma techniques,and used for the preparation of enzymatic reactor.Plasma oxidation for PDMS en-abled the channel wall of microfluidics to present a layer of silanol(SiOH)groups.These SiOH groups as anchorsonto the microchannel wall were linked covalently with the hydroxy groups of trypsin-encapsulated sol matrix.As aresult,the leakage of sol-gel matrix from the microchannel was effectively prevented.On-line protein analysis wasperformed with the microfluidic enzymatic reactor by attachment of stainless steel tubing electrode and replaceabletip.The success of trypsin encapsulation was investigated by capillary electrophoresis(CE)detection,and MALDITOF and ESI MS analysis.The lab-made device provided excellent extent of digestion even at the fast flow rate of7.0 μL/min with very short residence time of ca.2 s.In addition,the encapsulated trypsin exhibits increased stabil-ity even after continuous use.These features are the most requisite for high-throughput protein identification.     

Analysis of variations in the contents of steroidal saponins in Fructus Tribuli during stir-frying treatment

Just as natural saponins transform into aglycones, secondary glycosides and their derivatives using biotransformation technology, steroidal saponins may also undergo similar transformation after stir-frying. The purpose of this study was to elucidate the variations and the reasons for these variations in the contents of steroidal saponins in Fructus Tribuli (FT) during a stir-frying treatment. Stir-fried FT was processed in different time–temperature conditions. An UHPLC–MS/MS method was established and fully validated for quantitative analysis. In addition, the simulation processing products of tribuluside A, terrestroside B, terrestrosin K, terrestrosin D and 25R-tribulosin were determined by qualitative analysis using UHPLC–Q-TOF–MS. The established UHPLC–MS/MS method provides a rapid, flexible, and reliable method for the quality assessment of FT. The present study revealed that furostanol saponins with a C22-OH group could transform into corresponding furostanol saponins with a C-20–C-22 double bond (FSDB) via dehydroxylation. Additionally, FSDB could be successively converted into its secondary glycosides via a deglycosylation reaction. The transformation of spirostanol saponins into corresponding aglycones via deglycosylation led to a decrease in spirostanol saponins and an increase in aglycones. The results of this research provided scientific evidence of variation and structural transformation among steroidal saponins. These findings might be helpful for elucidating the processing mechanism of FT.     

Characterization of ginsenoside compound K metabolites in rat urine and feces by ultra‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Ginsenoside compound K (CK) is an active metabolite of ginsenoside and has been shown to have ameliorative property in various diseases. However, the detailed in vivo metabolism of this compound has rarely been reported. In the present study, a method using liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter and MS/MS scanning, was developed to detect and characterize the metabolites of CK in rat urine and feces. After oral administration of CK at a dose of 50 mg/kg, urine and feces were collected for a period of time and subjected to a series of pretreatment. A total of 12 metabolites were tentatively or conclusively identified, comprising 11 phase I metabolites and a phase II metabolite. Metabolic pathways of CK has been proposed, including oxidation, deglycosylation, deglycosylation with sequential oxidation and dehydrogenation and deglycosylation with sequential glucuronidation. Relative quantitative analyses suggested that deglycosylation was the main metabolic pathway. The result could offer insights for better understanding of the mechanism of its pharmacological activities.     

A facile and sensitive HPLC–MS/MS method for quantification of 3,3′-diindolylmethane in plasma samples

Indole-3-carbinol is the subject of ongoing biomedical research owing to its potential antiatherogenic, anticarcinogenic and antioxidant effects. The antitumor properties are mainly associated with its major metabolite, i.e. 3,3′-diindolylmethane (DIM). Typically, the biological activity of the chemical compound is manifested in the ng/ml concentration range. Consequently, the development of highly sensitive analytical methods to determine DIM in various biological samples is an urgent issue. In this study, an HPLC–MS/MS method was established for the quantification of DIM in human plasma. The developed method was validated according to the European Medicines Agency guidelines. Sensitivity, selectivity, accuracy and precision were good, allowing DIM quantification in the concentration range of 5–500 ng/ml. The limit of detection and the lower limit of quantification were 1 and 5 ng/ml, respectively. 4-Methoxy-1-methylindole was used as an internal standard (IS). The analytes were extracted from the human plasma by the acetonitrile-induced protein precipitation method with the addition of 3 mol/L ammonium sulfate as a salting-out agent, which is a facile and efficient approach for high-throughput bioanalysis. The chromatographic separation was performed on the Synergi Fusion-RP C₁₈ column (50 × 2.0 mm, 4 μm, 80 Å) under isocratic elution at 40°C. The mobile phase consisting of acetonitrile and water (0.1% formic acid; 85:15, v/v) was delivered at a flow rate of 0.20 ml/min. DIM and the IS were eluted at 2.36 ± 0.04 and 2.43 ± 0.03 min, respectively. The total analysis time was 3.20 min. Atmospheric pressure chemical ionization was carried out using multiple reaction monitoring in the positive polarity mode. The ion transitions were set to m/z 247.1 → 130.1 (DIM) and 162.1 → 147.1 (IS). The method was successfully applied to the analysis of plasma samples after a single oral administration of the Indinol® Forto drug (200 mg) to healthy female Russian volunteers. Also, the developed method was used for the analysis of rabbit plasma samples after a single oral dose of DIM (20 mg/kg).     

Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.     

A Stable Isotopic Pre-digestion Labeling Method for Protein Quantitative Analysis Using Matrix-assisted Laser Desorption/ionization Mass Spectrometry

As an extension of our previous work, here a strategy was demonstrated for protein identification and quantification analyses utilizing a combination of stable isotope chemical labeling with subsequent denaturation, enzymatic digestion and matrix assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). Using [d₀]‐ and [d₆]‐4,6‐dimethoxy‐2‐(methylsulfonyl)pyrimidine ([d₀]‐/[d₆]‐DMMSP), stable isotopic labels were incorporated before digestion. The comparative samples were combined before labeling after digestion, thus biases resulting from differences in sample digestion were avoided and the higher accuracy of quantification could be attained. The labeling was spatial‐selective to particular residues of cysteine, lysine, and tyrosine before denaturation, which could lead to a better universality of the strategy for cysteine‐free proteins. In addition, some lysine residues were blocked after labeling, the partly destroyed recognition sites could simplify the trypsin hydrolysates and hence facilitate the MS complexity. Together, our one‐step labeling strategy combined several desirable properties such as spatial‐selective labeling, reliability of quantitative results, simplification of analysis of complex systems and direct analysis with minimum sample handling. Our results demonstrate the usefulness of the method for analyzing lysozyme in egg white. The method was expected to provide a new powerful tool for comparative proteome research.     

Capillary electrophoresis and ultra-high-performance liquid chromatography methods in clinical monitoring of creatinine in human urine: A comparative study

Creatinine is an important diagnostic marker and is also used as a standardization tool for the quantitative evaluation of exogenous/endogenous substances in urine. This study aimed at evaluating and comparing three analytical approaches, based on hyphenations of different separation [two-dimensional capillary isotachophoresis (CITP–CITP), capillary zone electrophoresis (CZE), ultra-high-performance liquid chromatography (UHPLC)] and detection [conductivity (CD), ultraviolet (UV), tandem mass spectrometry (MS/MS)] techniques, for their ability to provide reliable clinical data along with their suitability for the routine clinical use (cost, simplicity, sample throughput). The developed UHPLC–MS/MS, CITP–CITP–CD, and CZE–UV methods were characterized by favorable performance parameters, such as linearity (r ˃ 0.99), precision (relative standard deviation, 0.22–2.97% for the creatinine position in analytical profiles), and recovery (87.1–115.1%). Clinical data, obtained from the analysis of 24 human urine samples by a reference enzymatic method, were comparable with those obtained by the tested methods (Passing–Bablok regression and Bland–Altman analysis), approving their usefulness for the routine clinical use. In this context, the UHPLC–MS/MS method provides benefits of enhanced orthogonality/accuracy and high sample throughput (threefold shorter total analysis times than the CE methods), whereas advantages of the CE methods for routine labs are simplicity and low cost of both the instrumentation and measurements.     

基于UPLC-Q-Orbitrap MS/MS技术研究酸枣仁发酵过程中的化学成分转化

利用超高效液相色谱-四极杆-静电场轨道阱串联质谱（UPLC-Q-Orbitrap MS/MS）研究了酸枣仁经茯苓发酵前后的主要化学成分变化，初步探讨了茯苓对酸枣仁发酵的转化规律.鉴定了发酵产物中24种化学成分，整合多元统计方法对酸枣仁发酵0，7和14 d后的差异性化学成分进行研究，采用正交偏最小二乘分析找到7个差异成分.采用t-检验对24种化学成分的相对含量进行分析；并分析了酸枣仁中黄酮苷、皂苷类成分的代谢反应途径.通过茯苓发酵，酸枣仁中化学成分主要发生了脱酰基和脱糖基等水解反应.为酸枣仁发酵产物的化学成分检测提供了快速、直观且准确的方法，为酸枣仁-茯苓共发酵产物的深入开发与利用提供了科学依据.     

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography–tandem mass spectrometry (LC‐MS/MS) was developed. This assay represents the first LC‐MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3‐atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/mL and 10 nm for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3–900 ng/mL and 10 nm to 10 µm for human plasma and cellular samples, respectively (r² > 0.999). The intra‐ and inter‐day assay accuracy and precision were evaluated using quality control samples at three different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect and recovery were also successfully demonstrated. The present assay is superior to previously published LC‐MS and LC‐MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. Copyright © 2012 John Wiley & Sons, Ltd.     

In-gel deglycosylation of sodiumdodecyl sulfate polyacrylamide gel electrophoresis-separated glycoproteins for carbohydrate estimation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Mass determination by mass spectrometric methods (electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS)) of sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)-separated proteins is a well known procedure and reliable protocols are available. In our efforts to use the established methods to determine the molecular mass of the disulfide bridged, heterodimeric glycoprotein GP3 and to determine the carbohydrate content of each protein subunit we developed an in-gel chemical deglycosylation method. For this purpose we established experimental conditions that allow maximum extraction of the high molecular mass protein subunits and developed a routine method to apply the HF-pyridine deglycosylation protocol to proteins isolated from polyacrylamide gel pieces. The novel protocol and extraction procedure described can be used to analyze O-glycosylated proteins up to 150 kDa after SDS-PAGE separation.