Proteomic profiling of human pleural effusion using two-dimensional nano liquid chromatography tandem mass spectrometry

Pleural effusion, an accumulation of pleural fluid, contains proteins originated from plasma filtrate and, especially when tissues are damaged, parenchyma interstitial spaces of lungs and/or other organs. This study details protein profiles in human pleural effusion from 43 lung adenocarcinoma patients by a two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system. The experimental results revealed the identification of 1415 unique proteins from human pleural effusion. Among these 124 proteins identified with higher confidence levels, some proteins have not been reported in plasma and may represent proteins specifically present in pleural effusion. These proteins are valuable for mass identification of differentially expressed proteins involved in proteomics database and screening biomarker to further study in human lung adenocarcinoma. The significance of the use of proteomics analysis of human pleural fluid for the search of new lung cancer marker proteins, and for their simultaneous display and analysis in patients suffering from lung disorders has been examined.     

Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry

The tissue distribution of folate in its numerous coenzyme forms may influence the development of disease at different sites. For instance, the susceptibility of human colonic mucosa to localized folate deficiency may predispose to the development of colorectal cancer. We report a sensitive and robust ultra high-performance liquid chromatography (UHPLC) tandem mass spectrometry method for quantifying tissue H₄folate, 5-CH₃-H₄folate, 5-CHO-H₄folate, folic acid, and 5,10-CH⁺-H₄folate concentration. Human colonic mucosa (20–100 mg) was extracted using lipase and conjugase enzyme digestion. Rapid separation of analytes was achieved on a UHPLC 1.9-μm C18 column over 7 min. Accurate quantitation was performed using stable isotopically labeled (¹³C₅) internal standards. The instrument response was linear over physiological concentrations of tissue folate (R² > 0.99). Limits of detection and quantitation were less than 20 and 30 fmol on column, respectively, and within- and between-run imprecision values were 6–16%. In colonic mucosal samples from 73 individuals, the average molar distribution of folate coenzymes was 58% 5-CH₃-H₄folate, 20% H₄folate, 18% formyl-H₄folate (sum of 5-CHO-H₄folate and 5,10-CH⁺-H₄folate), and 4% folic acid. This assay would be useful in characterizing folate distribution in human and animal tissues as well as the role of deregulated folate homeostasis on disease pathogenesis.     

Bioanalysis in clinical development of tasquinimod using liquid chromatography/tandem mass spectrometry

Tasquinimod (ABR-215050) is an oral drug in clinical development for treatment of patients with castrate resistant prostate cancer. This paper describes a method for the determination of tasquinimod in human plasma. The method is based on liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) using stable isotope labeled tasquinimod as internal standard (IS). The plasma samples were processed by protein precipitation using acidic acetonitrile containing the IS. The precipitated samples were centrifuged and the supernatant was injected directly into the LC-MS/MS system. Chromatographic separation was performed on a reversed phase column using fast gradient elution, with a total run cycle time of 4 min. The method was validated with respect to accuracy, precision, dynamic range, lower limit of quantification, selectivity and robustness. Furthermore, the stability of tasquinimod in spiked plasma, in processed extracts and in incurred samples was thoroughly studied. The method was validated in the range of 1.0-2400 nmol/L, defining the lower and upper limits of quantification. The repeatability, reproducibility and overall bias were 1.5-7.1%, 3.5-7.4%, and 1.3-4.7%, respectively, in the range of 1-2000 nmol/L. Excellent selectivity was demonstrated in the validation, as well as in study samples from both healthy volunteers and cancer patients. Robustness was demonstrated by the calculated carry-over as low as 0.06%, and by an incurred sample reproducibility (ISR) experiment where 97% of the reanalyzed samples fulfilled the acceptance criteria of 20% deviation from initial analysis result. Also, tasquinimod was found to be stable in all investigated matrices, including in incurred samples. In an incurred sample stability (ISS) investigation, tasquinimod was demonstrated to be stable for 24 months, and 97% of the reanalyzed samples were within 20% from the initial analysis result. In conclusion, the method was demonstrated to be accurate, precise, robust and reliable for the determination of tasquinimod. The method was successfully used in several clinical studies for the support of pharmacokinetic and pharmacodynamic evaluations.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

Determination of benzimidazole residues using liquid chromatography and tandem mass spectrometry

The determination of residues of benzimidazole using liquid chromatography and tandem mass spectrometry (LC–MS–MS) with ion spray ionization is described. Swine muscle tissue was spiked with a mixture of fifteen benzimidazoles, including metabolites of fenbendazole and albendazole. As clean-up procedure, an ethyl acetate extraction followed by solid-phase extraction on styrol-divinyl-benzene cartridge was used. The evaluation was performed by selecting the characteristic product ions for the benzimidazoles and using multiple reaction mode. 2-n-Butylmercaptobenzimidazole was used as internal standard. Blank muscle samples were fortified in the concentration range of 1–22 μg/kg. The limits of detection were below 6 μg/kg and the limits of quantification for most benzimidazoles were below 10 μg/kg. The matrix effect was checked using spiked muscle tissues of cattle and sheep as well as liver of cattle. Practical application will be shown by incurred egg material from laying hens treated with flubendazole. The recovery of the clean-up was mostly above 50% in muscle tissue and 70% in egg yolk.     

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

In the frame of protein identification from mouse adipose tissue, two strategies were compared for the offline elution of peptides from a strong cation exchange (SCX) column in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) analyses. First, the salt gradient (using K⁺ as displacing agent) was evaluated from 25 to 500 mM KCl. Then, a less investigated elution mode using a pH gradient (using citric acid and ammonium hydroxide) was carried out from pH 2.5 to 9.0. Equal amounts of peptide digest derived from mouse adipose tissue were loaded onto the SCX column and fractionated according to the two approaches. A total of 15 fractions were collected in two independent experiments for each SCX elution strategy. Then, each fraction was analyzed on a nanoLC–MS/MS platform equipped with a column-switching unit for desalting and enrichment. No substantial differences in peptide quality characteristics (molecular weight, isoelectric point, or GRAVY [grand average of hydropathicity] index distributions) were observed between the two datasets. The pH gradient approach was found to be superior, with 27.5% more unique peptide identifications and 10% more distinct protein identifications compared with the salt-based elution method. In conclusion, our data imply that the pH gradient SCX fractionation is more desirable for proteomics analysis of entire adipose tissue.     

Fully automatable two-dimensional reversed-phase capillary liquid chromatography with online tandem mass spectrometry for shotgun proteomics

Herein, we describe the development of a fully automatable technology that features online coupling of high‐pH RP separation with conventional low‐pH RP separation in a two‐dimensional capillary liquid chromatography (2‐D LC) system for shotgun proteomics analyses. The complete analysis comprises 13 separation cycles, each involving transfer of the eluate from the first‐dimension, high‐pH RP separation onto the second RP dimension for further separation. The solvent strength increases across the 13 fractions (cycles) to elute all peptides for further resolution on the second‐dimension, low‐pH RP separation, each under identical gradient‐elution conditions. The total run time per analysis is 52 h. In triplicate analyses of a lysate of mouse embryonic fibroblasts, we used this technology to identify 2431 non‐redundant proteins, of which 50% were observed in all three replicates. A comparison of RP‐RP 2‐D LC and strong cation exchange‐RP 2‐D LC analyses reveals that the two technologies identify primarily different peptides, thereby underscoring the differences in their separation chemistries.     

Identification of candidate biomarker proteins released by human endometrial and cervical cancer cells using two-dimensional liquid chromatography/tandem mass spectrometry

Candidate biomarker proteins, including chaperonin 10 and pyruvate kinase, previously discovered and identified using mass-tagging reagents with multidimensional liquid chromatography and tandem mass spectrometry (DeSouza, L.; et al. J. Proteome Res. 2005, 4, 377-386) have been identified in serum-free media of cultured endometrial cancer (KLE and HEC-1-A) and cervical cancer (HeLa) cells. These and other cancer-associated proteins were released by the cultured cells within 24 h of growth. A total of 203 proteins from the KLE cells, 86 from HEC-1-A, and 161 from HeLa are reported.     

Quantitative method for biomarkers of collagen degradation using liquid chromatography tandem mass spectrometry

Preclinical efficacy testing commonly involves studies that require considerable resources and time. One valuable tool in this endeavor is the characterization of relevant biomarkers. A method has been developed for the simultaneous determination of collagen biomarker candidates as an instrument in screening compounds for efficacy. Two potential candidates, the 3-hydroxypyridinium crosslinks pyridinoline and deoxypyridinoline, were selected for analysis in collagen degradation models. Tissue or urine samples were collected, prepared, and quantitated for the biomarkers using spiked calibration curves and liquid chromatography tandem mass spectrometry. The development of a quick and simple assay method would allow us to increase the chances for success in efficacy screening by eliminating compounds with poor biomarker profiles. The method proposed here appears to be more selective, convenient, precise (generally <10% RSD), accurate (generally <10% RE), and sensitive relative to previously established methodology.     

Analysis of mammalian sphingolipids by liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS)

Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or "sphingolipidomic" methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices.     

Prenatal diagnosis for organic acid disorders using two mass spectrometric methods, gas chromatography mass spectrometry and tandem mass spectrometry

We performed prenatal diagnosis of organic acid disorders using two mass spectrometric methods; gas chromatography mass spectrometry (GC/MS) and tandem mass spectrometry (ESI/MS/MS). Of 28 cases whose amniotic fluid was tested, 11 cases were diagnosed as "affected". All cases whose samples were diagnosed as "unaffected" were confirmed to have no symptoms or abnormalities in urinary organic acid analysis after birth. Of the 11 "affected" cases, two cases were missed by ESI/MS/MS but not by GC/MS. When the stability of metabolites in amniotic fluid was checked, it was found that acylcarnitines degraded in one week at room temperature, whereas organic acids such as methylmalonate or methylcitrate were stable for at least 14 days. Prenatal diagnosis by analysis using simultaneous two or more methods may be more reliable, though attention should be paid to sample transportation conditions.     

Automated, on-line two-dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests

Evaluation of cellular processes and their changes at the level of protein expression and post-translational modifications may allow identification of novel proteins and the mechanisms involved in pathogenic processes. However, the number of proteins and, after tryptic digestion, of peptides from a single cell can be tremendously high. Separation and analysis of such complex peptide mixtures can be performed using multidimensional separation techniques such as two-dimensional gel electrophoresis or two-dimensional-high-performance liquid chromatography (2-D-HPLC). The aim of this work was to establish a fully automated on-line 2-D-HPLC separation method with column switching for the separation of complex tryptic digests. A model mixture of five proteins as well as a nuclear matrix protein sample were digested with trypsin and separated using a strong cation exchange (SCX) column in the first dimension and nano reversed phase in the second dimension. Separated peptides were detected using an ion trap mass spectrometer. The advantages of this new fully automated method are rapid sample loading, the possibility of injecting large volumes and no introduction of salt into the mass spectrometer. Furthermore, column switching allows the independent control and optimization of the two dimensions independently.     

Analysis of murine natural killer cell microsomal proteins using two-dimensional liquid chromatography coupled to tandem electrospray ionization mass spectrometry

This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (microRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membrane-spanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11,967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins.     

Quantitative urine amino acid analysis using liquid chromatography tandem mass spectrometry and aTRAQ reagents

Ion-exchange chromatography (IEC) is the most widely used method for amino acid analysis in physiological fluids because it provides excellent separation and reproducibility, with minimal sample preparation. The disadvantage, however, is the long analysis time needed to chromatographically resolve all the amino acids. To overcome this limitation, we evaluated a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method, which utilizes aTRAQ reagents, for amino acid analysis in urine. aTRAQ reagents tag the primary and secondary amino groups of amino acids. Internal standards for each amino acid are also labeled with a modified aTRAQ tag and are used for quantification. Separation and identification of the amino acids is achieved by liquid chromatography tandem mass spectrometry using retention times and mass transitions, unique to each amino acid, as identifiers. The run time, injection-to-injection, is 25 min, with all amino acids eluting within the first 12 min. This method has a limit of quantification (LOQ) of 1 μmol/L, and is linear up to 1000 μmol/L for most amino acids. The Coefficient of Variation (CV) was less than 20% for all amino acids throughout the linear range. Method comparison demonstrated concordance between IEC and LC-MS/MS and clinical performance was assessed by analysis of samples from patients with known conditions affecting urinary amino acid excretion. Reference intervals established for this method were also concordant with reference intervals obtained with IEC. Overall, aTRAQ reagents used in conjunction with LC-MS/MS should be considered a comparable alternative to IEC. The most attractive features of this methodology are the decreased run time and increased specificity.     

Quantitative assay for six potential breast cancer biomarker peptides in human serum by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify several possible breast cancer peptide biomarkers in human serum has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The peptides include bradykinin, Hyp³-bradykinin, des-Arg⁹-bradykinin and fragments of fibrinogen α-chain (Fib-α_[605–629]), inter-α-trypsin inhibitor heavy chain 4 (ITIH_4[666–687]) and complement component 4a (C4a_{[1337–1350]}). Ile¹³-ITIH_4[666–687], d20-C4a_{[1337–1350]} and Sar-D-Phe⁸-des-Arg⁹-bradykinin were used as internal standards. Bovine plasma, with 2 mM captopril and 2 mM d-l-mercaptoethanol-3-guanidino-ethylthiopropanoic acid (MEGETPA) to prevent rapid degradation of the bradykinins, was used as analyte-free matrix. Recoveries for solid-phase extraction (SPE) on mixed-mode weak cation exchange sorbents were between 62 and 90%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with a heated electrospray source (H-ESI), operating in the positive ion-mode, was used for detection. The assay was fully validated and stabilities of the peptides were extensively explored. Bradykinin (10–500 ng/ml), Hyp³-bradykinin (4–200 ng/ml), des-Arg⁹-bradykinin (2–100 ng/ml), Fib-α_[605–629] (120–3000 ng/ml), ITIH_4[666–687] (0.4–10 ng/ml) and C4a_{[1337–1350]} (1–25 ng/ml) were simultaneously quantified with deviations from the nominal concentrations below 22% and intra- and inter-assay precisions below 15 and 20%, respectively, for all peptides at all concentrations. The method has been successfully applied to several serum samples from breast cancer patients and matched controls.     

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography–electrospray ionization–tandem mass spectrometry (HILIC–ESI–MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5 pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC–pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.     

Pharmacokinetics of ergosterol in rats using rapid resolution liquid chromatography-atmospheric pressure chemical ionization multi-stage tandem mass spectrometry and rapid resolution liquid chromatography/tandem mass spectrometry

Rapid resolution liquid chromatography/tandem multi-stage mass spectrometry (RRLC-MS(n)) and rapid resolution liquid chromatography/tandem mass spectrometry (RRLC/MS/MS) methods were developed for the identification and quantification of ergosterol and its metabolites from rat plasma, urine and faeces. Two metabolites (ERG1 and ERG2) were identified by RRLC/MS(n). The concentrations of the ergosterol were determined by RRLC/MS/MS. The separation was performed on an Agilent Zorbax SB-C18 with the mobile phase consisting of methanol and water (containing 0.1% formic acid). The detection was carried out by means of atmospheric pressure chemical ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 7-2000, 6-2000 and 8-7500 ng/mL for plasma, urine and faecal homogenate, respectively. The intra- and inter-day precision values (RSD) were below 10%. The method was applied to the pharmacokinetic properties and elimination pathway of ergosterol in rats.