Bias reduction in the quantitative analysis of a target analyte present in a limited quantity in human plasma using dual-mode heart-cutting two-dimensional liquid chromatography coupled with isotope dilution mass spectrometry.

Dual-mode heart-cutting two-dimensional liquid chromatography (DMHC 2D-LC) was applied to isotope dilution mass spectrometry (IDMS) to reduce the bias in the quantitative analysis of a target analyte present in a limited quantity in human plasma. Based on a Waters I-Class LC system, the DMHC 2D-LC system was operated in one- and two-dimensional modes to facilitate the determination of heart-cutting time and the efficient trapping of the target LC eluate. Experiments to determine the feasibility of coupling with IDMS were performed with triple quadrupole mass spectrometry using folic acid standards and/or 13 C5 -folic acid. To validate the performance of the DMHC 2D-LC/IDMS system on a complex sample, human plasma was analyzed for folic acid and the result was compared with that obtained using conventional single-column LC. The total run time of the DMHC 2D-LC system was 20 min, the same as that of the single-column LC system. The peak profile of the spiked 13 C5 -folic acid obtained with single-column LC/MS was affected by matrix effects, but resolved with DMHC 2D-LC/MS, thus improving the accuracy of the analysis. The DMHC 2D-LC/IDMS system showed reliable performance in analyzing the target analyte in human plasma, eliminating matrix effects and saving analysis time.

Microscale depletion of high abundance proteins in human biofluids using IgY14 immunoaffinity resin: analysis of human plasma and cerebrospinal fluid.

Removal of highly abundant proteins in plasma is often carried out using immunoaffinity depletion to extend the dynamic range of measurements to lower abundance species. While commercial depletion columns are available for this purpose, they generally are not applicable to limited sample quantities (<20 µL) due to low yields stemming from losses caused by nonspecific binding to the column matrix and concentration of large eluent volumes. Additionally, the cost of the depletion media can be prohibitive for larger-scale studies. Modern LC-MS instrumentation provides the sensitivity necessary to scale-down depletion methods with minimal sacrifice to proteome coverage, which makes smaller volume depletion columns desirable for maximizing sample recovery when samples are limited, as well as for reducing the expense of large-scale studies. We characterized the performance of a 346 µL column volume microscale depletion system, using four different flow rates to determine the most effective depletion conditions for ∼6-µL injections of human plasma proteins and then evaluated depletion reproducibility at the optimum flow rate condition. Depletion of plasma using a commercial 10-mL depletion column served as the control. Results showed depletion efficiency of the microscale column increased as flow rate decreased, and that our microdepletion was reproducible. In an initial application, a 600-µL sample of human cerebrospinal fluid (CSF) pooled from multiple sclerosis patients was depleted and then analyzed using reversed phase liquid chromatography-mass spectrometry to demonstrate the utility of the system for this important biofluid where sample quantities are more commonly limited.

Certification and long-term stability monitoring of low-content folic acid and 5-methyltetrahydrofolate in human plasma certified reference material by isotope dilution ultra-high performance liquid chromatography/tandem mass spectrometry.

A reliable measurement of blood folate levels is necessary in the clinical field to prevent serious diseases such as cognitive impairment and neural tube defects. Herein, the certification of the low-content folic acid (FA) and 5-methyltetrahydrofolate (5-Me-THF) in human plasma certified reference material (KRISS CRM 111-01-018) was performed. A human plasma pool obtained from the Korean Red Cross was used as a CRM candidate. The certification of the human plasma CRM was performed by isotope dilution ultra-performance liquid chromatography/tandem mass spectrometry. Two-dimensional liquid chromatography was employed to confirm the validity of the analytical method for FA due to the susceptibility of FA to matrix effects because of its limited quantity. The CRM stability was evaluated at -20 °C for 2 months and at -70 °C for up to 12 months to determine the certified value of the CRM. The certified value of the CRM was (84.6 ± 4.3) ng kg-1 and (5.80 ± 0.47) µg kg-1 for FA and 5-Me-THF, respectively. The homogeneity of the CRM was 1.64% and 3.10% for FA and 5-Me-THF, respectively. Further long-term stability assessments were conducted, indicating that the CRM remains valid for at least 58 months at -70 °C for FA and 48 months for 5-Me-THF. Compared to other blood-based CRMs, this CRM has lower folate levels, making it helpful in establishing analytical methods for a broader range of folate levels.

A serum protein profile predictive of the resistance to neoadjuvant chemotherapy in advanced breast cancers.

Prediction of the responses to neoadjuvant chemotherapy (NACT) can improve the treatment of patients with advanced breast cancer. Genes and proteins predictive of chemoresistance have been extensively studied in breast cancer tissues. However, noninvasive serum biomarkers capable of such prediction have been rarely exploited. Here, we performed profiling of N-glycosylated proteins in serum from fifteen advanced breast cancer patients (ten patients sensitive to and five patients resistant to NACT) to discover serum biomarkers of chemoresistance using a label-free liquid chromatography-tandem MS method. By performing a series of statistical analyses of the proteomic data, we selected thirteen biomarker candidates and tested their differential serum levels by Western blotting in 13 independent samples (eight patients sensitive to and five patients resistant to NACT). Among the candidates, we then selected the final set of six potential serum biomarkers (AHSG, APOB, C3, C9, CP, and ORM1) whose differential expression was confirmed in the independent samples. Finally, we demonstrated that a multivariate classification model using the six proteins could predict responses to NACT and further predict relapse-free survival of patients. In summary, global N-glycoproteome profile in serum revealed a protein pattern predictive of the responses to NACT, which can be further validated in large clinical studies.

Fully automated multifunctional ultrahigh pressure liquid chromatography system for advanced proteome analyses.

A multifunctional liquid chromatography system that performs 1-dimensional, 2-dimensional (strong cation exchange/reverse phase liquid chromatography or SCX/RPLC) separations and online phosphopeptide enrichment using a single binary nanoflow pump has been developed. With a simple operation of a function selection valve equipped with a SCX column and a TiO2 (titanium dioxide) column, a fully automated selection of three different experiment modes was achieved. Because the current system uses essentially the same solvent flow paths, the same trap column, and the same separation column for reverse-phase separation of 1D, 2D, and online phosphopeptides enrichment experiments, the elution time information obtained from these experiments is in excellent agreement, which facilitates correlating peptide information from different experiments. The final reverse-phase separation of the three experiments is completely decoupled from all of the function selection processes; thereby salts or acids from SCX or TiO2 column do not affect the efficiency of the reverse-phase separation.

The effect and potential of using a temperature controlled separation column with ultra-high pressure microcapillary liquid chromatography/tandem mass spectrometry on proteomic analysis.

The microcapillary liquid chromatography (µLC)/tandem mass spectrometry (MS/MS) system has become a prevailing analytical platform in proteomics. Typical proteomic studies aimed at proteome-wide identification of peptides and proteins rely heavily on producing an accurate and reproducible solvent-composition gradient throughout microcapillary separation columns to improve LC separation. With the recent advent of targeted proteomic approaches utilizing the LC retention time as a physicochemical parameter for peptides, high reproducibility of LC separation additionally becomes an important factor. In this study, column temperature elevation is utilized to improve reproducibility and separation efficiency of the µLC-MS/MS system. The simple incorporation of a semi-rigid gas line heater allowed precise control of the temperature of microcapillary columns longer than 70 cm, up to 60 °C. Tryptic enolase peptides were used as a standard sample to evaluate the effect of the controlled temperature elevation on the peptide separation efficiency and reproducibility. In addition to the increased reproducibility in peptide elution time due to the controlled column temperature, the temperature elevation resulted in a decrease in the column operation pressure, which, in turn, allowed a higher solvent flow-rate to be employed using the same LC pumps, leading to further improvements in the performance of µLC systems.

Highly sensitive analytical method for the accurate determination of 5-methyltetrahydrofolic acid monoglutamate in various volumes of human plasma using isotope dilution ultra-high performance liquid chromatography-mass spectrometry.

One predominant and bioactive folate vitamer circulating in the blood is 5-methyltetrahydrofolate (5-Me-THF). In this study, a method for the accurate determination of 5-Me-THF in human plasma samples of various volumes was established using isotope dilution ultra-high performance liquid chromatography-mass spectrometry (ID-UPLC-MS). For this purpose, 500 µL of homogeneous human plasma was initially employed, and the 5-Me-THF and the 13C5-5-Me-THF standard solutions prepared using 1% ascorbic acid in water gave the calibration solution and spiking sample. The desired amount of 13C5-5-Me-THF standard solution was spiked into the sample followed by sample pretreatment. The method was validated for its repeatability, reproducibility, recovery, and limits of detection and quantification. Subsequently, it was applied to smaller volumes of human plasma samples (i.e., 50 and 10 µL), the results of which corresponded well with those obtained using 500 µL. The feasibility of the method was further confirmed using 10 µL of a standard reference material, SRM 3949, which is a human serum sample containing three different levels of 5-Me-THF. The established ID-UPLC-MS method was successfully applied to various volumes of human plasma or serum ranging from 500 to 10 µL, which exhibited particularly good sensitivity in addition to reliable results for the quantification of 5-Me-THF. Our method therefore expands on the ability to obtain accurate quantitative results for 5-Me-THF using small volumes of blood.

Mitochondrial oxidative phosphorylation system is recruited to detergent-resistant lipid rafts during myogenesis.

Since detergent-resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent-resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface-biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH-dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.

Integrated post-experiment monoisotopic mass refinement: an integrated approach to accurately assign monoisotopic precursor masses to tandem mass spectrometric data.

Accurate assignment of monoisotopic precursor masses to tandem mass spectrometric (MS/MS) data is a fundamental and critically important step for successful peptide identifications in mass spectrometry based proteomics. Here we describe an integrated approach that combines three previously reported methods of treating MS/MS data for precursor mass refinement. This combined method, "integrated post-experiment monoisotopic mass refinement" (iPE-MMR), integrates steps (1) generation of refined MS/MS data by DeconMSn; (2) additional refinement of the resultant MS/MS data by a modified version of PE-MMR; and (3) elimination of systematic errors of precursor masses using DtaRefinery. iPE-MMR is the first method that utilizes all MS information from multiple MS scans of a precursor ion including multiple charge states, in an MS scan, to determine precursor mass. With the combination of these methods, iPE-MMR increases sensitivity in peptide identification and provides increased accuracy when applied to complex high-throughput proteomics data.

Postexperiment monoisotopic mass filtering and refinement (PE-MMR) of tandem mass spectrometric data increases accuracy of peptide identification in LC/MS/MS.

Methods for treating MS/MS data to achieve accurate peptide identification are currently the subject of much research activity. In this study we describe a new method for filtering MS/MS data and refining precursor masses that provides highly accurate analyses of massive sets of proteomics data. This method, coined "postexperiment monoisotopic mass filtering and refinement" (PE-MMR), consists of several data processing steps: 1) generation of lists of all monoisotopic masses observed in a whole LC/MS experiment, 2) clusterization of monoisotopic masses of a peptide into unique mass classes (UMCs) based on their masses and LC elution times, 3) matching the precursor masses of the MS/MS data to a representative mass of a UMC, and 4) filtration of the MS/MS data based on the presence of corresponding monoisotopic masses and refinement of the precursor ion masses by the UMC mass. PE-MMR increases the throughput of proteomics data analysis, by efficiently removing "garbage" MS/MS data prior to database searching, and improves the mass measurement accuracies (i.e. 0.05 +/- 1.49 ppm for yeast data (from 4.46 +/- 2.81 ppm) and 0.03 +/- 3.41 ppm for glycopeptide data (from 4.8 +/- 7.4 ppm)) for an increased number of identified peptides. In proteomics analyses of glycopeptide-enriched samples, PE-MMR processing greatly reduces the degree of false glycopeptide identification by correctly assigning the monoisotopic masses for the precursor ions prior to database searching. By applying this technique to analyses of proteome samples of varying complexities, we demonstrate herein that PE-MMR is an effective and accurate method for treating massive sets of proteomics data.

Development of an infant formula certified reference material for the analysis of organic nutrients.

Infant formula certified reference material (CRM, KRISS CRM 108-02-003) were developed for the analysis of organic nutrients. The CRM is a milk-based infant formula powder, packaged at 14 g per unit. Ten thousand units were prepared and stored at -70 °C. For the certification of each nutrient, ten units were analyzed for simultaneous value-assignment and homogeneity test. Analytical methods used were isotope dilution mass spectrometry (IDMS) based on liquid chromatography mass spectrometer (LC/MS) or gas chromatography mass spectrometer (GC/MS) as higher-order reference methods.13 vitamins, 3 fatty acids, and total cholesterol were certified. The between-unit relative standard deviation of measurement results for each nutrient ranged 0.2% to 2.5%, showing very good homogeneity. The expanded relative uncertainties of the certified values ranged from 1% to 8%, indicating that they have higher-order metrological quality. The values of proximates (proteins, lipids, carbohydrates, water, and ash) were assigned through inter-laboratory comparisons.

Development of certified reference materials for accurate determination of fluoroquinolone antibiotics in chicken meat.

Certified reference materials (CRMs; KRISS CRM 108-03-003, 108-03-004) were developed for the accurate determination of fluoroquinolones (enrofloxacin and ciprofloxacin, respectively) in chicken meat. Two groups of chickens were cured with feeds containing enrofloxacin and ciprofloxacin, respectively. After slaughter, the thigh and breast meats were combined for the respective groups and the meat was freeze-dried, pulverized, sieved, and V-mixed. The final bulk material was bottled in 10g portions. For certification of the CRMs, isotope dilution-liquid chromatography/tandem mass spectrometry was used. The certified values of the CRMs were (19.06±0.86)mg/kg for enrofloxacin and (1.095±0.038)mg/kg for ciprofloxacin. The stabilities of the CRMs were monitored at -70°C for 12months, at -20°C for 2months, and at room temperature for 1month. Both CRM candidates were stable during the monitoring period for each temperature.

A putative early response of antifungal Bacillus lentimorbus WJ5 against the plant pathogenic fungus, Colletotrichum gloeosporioides, analyzed by a DNA microarray.

The global RNA transcription profiles of Bacillus lentimorbus WJ5 under an in vitro co-culture with Colletotrichum gloeosporioides were analyzed in order to study the antagonistic bacteria-fungi interactions. Using a filter membrane system, B. lentimorbus WJ5 was exposed to the spores of C. gloeosporioides at the late exponential stage. The transcription profiles of the B. lentimorbus WJ5, both with and without a challenge from C. gloeosporioides, were analyzed using custom DNA chips containing 2,000 genome fragments. A total of 337 genes were expressed, with 87 and 47 up- and down-regulated, respectively. Of these, 12 genes, which were involved in central carbon metabolisms, and 7 from minor catabolism were relatively highly up-regulated (> 10 fold) and down-regulated (< 0.2 fold), respectively. Nine genes, which were thought to be related to the antifungal activity, were also up-regulated, but their levels were not so high (2.0 - 9.7 folds). From the results, during the early stage of the co-culture of B. lentimorbus WJ5 and C. gloeosporioides, nutrient competition seemed to occur; therefore, the genes from central carbon metabolisms could be up-regulated, while those from minor catabolism could be down-regulated.

Plasma membrane calcium ATPase regulates bone mass by fine-tuning osteoclast differentiation and survival.

The precise regulation of Ca(2+) dynamics is crucial for proper differentiation and function of osteoclasts. Here we show the involvement of plasma membrane Ca(2+) ATPase (PMCA) isoforms 1 and 4 in osteoclastogenesis. In immature/undifferentiated cells, PMCAs inhibited receptor activator of NF-κB ligand-induced Ca(2+) oscillations and osteoclast differentiation in vitro. Interestingly, nuclear factor of activated T cell c1 (NFATc1) directly stimulated PMCA transcription, whereas the PMCA-mediated Ca(2+) efflux prevented NFATc1 activation, forming a negative regulatory loop. PMCA4 also had an anti-osteoclastogenic effect by reducing NO, which facilitates preosteoclast fusion. In addition to their role in immature cells, increased expression of PMCAs in mature osteoclasts prevented osteoclast apoptosis both in vitro and in vivo. Mice heterozygous for PMCA1 or null for PMCA4 showed an osteopenic phenotype with more osteoclasts on bone surface. Furthermore, PMCA4 expression levels correlated with peak bone mass in premenopausal women. Thus, our results suggest that PMCAs play important roles for the regulation of bone homeostasis in both mice and humans by modulating Ca(2+) signaling in osteoclasts.

Lyn inhibits osteoclast differentiation by interfering with PLCgamma1-mediated Ca2+ signaling.

Osteoclasts differentiate from macrophage-lineage cells to become specialized for bone resorption function. By a proteomics approach, we found that Lyn was down-regulated by the osteoclast differentiation factor, receptor activator of NF-kappaB ligand (RANKL). The forced reduction of Lyn caused a striking increase in the RANKL-induced PLCgamma1, Ca(2+), and NFATc1 responses during differentiation. These data suggest that Lyn plays a negative role in osteoclastogenesis by interfering with the PLCgamma1-mediated Ca(2+) signaling that leads to NFATc1 activation. Consistent with the in vitro results, in vivo injection of Lyn specific siRNA into mice calvariae provoked a fulminant bone resorption. Our study provides the first evidence of the involvement of Lyn in the negative regulation of osteoclastogenesis by RANKL.

The ubiquitin-mediated degradation of Jak1 modulates osteoclastogenesis by limiting interferon-beta-induced inhibitory signaling.

Interferons (IFNs) have been shown to negatively regulate osteoclastogenesis. In a proteomic study to assess protein expression during osteoclastogenesis, we discovered that the expression level of Jak1 was significantly decreased during the early stage of osteoclast differentiation from mouse bone marrow macrophages (BMMs) upon stimulation with receptor activator of nuclear factor kappaB ligand (RANKL). RANKL induced Jak1 ubiquitination, and a proteasome inhibitor MG132 efficiently blocked the RANKL-induced degradation of Jak1. The expression level of Jak1 correlated with the susceptibility of osteoclast precursors to the negative regulatory effects of IFN-beta on osteoclastogenesis, since preosteoclasts (pOCs) in which Jak1 expression is significantly reduced could proceed with osteoclastogenesis in the presence of IFN-beta. Forced down-regulation of Jak1 by small interfering RNA (siRNA) resulted in the efficient osteoclast differentiation of BMMs in the presence of inhibitory IFN-beta, while overexpression of Jak1 in pOCs elicited IFN-beta-dependent inhibition of osteoclastogenesis. Furthermore, we found that the IFN-beta-induced inhibition of osteoclastogenesis required STAT3 downstream of Jak1. These data suggest that the regulation of Jak1 expression during osteoclast differentiation might serve as an intrinsic mechanism that determines osteoclast lineage commitment by modulating the negative regulation by IFN-beta.

Ultrahigh-pressure dual online solid phase extraction/capillary reverse-phase liquid chromatography/tandem mass spectrometry (DO-SPE/cRPLC/MS/MS): a versatile separation platform for high-throughput and highly sensitive proteomic analyses.

Capillary RPLC/ESI-MS (cRPLC/ESI-MS) is one of the most powerful analytical tools for current proteomic research. The development of cRPLC techniques coupled online to a mass spectrometer has focused on increasing the separation efficiency, detection sensitivity, and throughput. Recently, the use of high-pressure (over 10,000 psi) LC systems that utilize long, small inner diameter capillary columns has gained much attention for proteomic analyses. In this study, we developed an ultrahigh-pressure dual online SPE/capillary RPLC (DO-SPE/cRPLC) system. This LC system employs two online SPE columns and two capillary columns (75 microm inner diameter x 1 m length) in a single separation system, and has a maximum operating pressure of 10,000 psi. This DO-SPE/cRPLC system is capable of providing high-resolution separation in addition to several other advantageous features, such as high reproducibility in terms of the LC retention time, rapid sample injection, online desalting, online sample enrichment of dilute samples, and increased throughput as a result of essentially removing the column equilibration time between successive experiments. We coupled the DO-SPE/cRPLC system online to a tandem mass spectrometer to allow high-throughput proteomic analyses. In this paper, we demonstrate the efficiency of this DO-SPE/cRPLC/MS/MS system by its use in the analyses of proteomic samples exhibiting different levels of complexity.

Photodissociation tandem mass spectrometry at 266 nm of an aliphatic peptide derivatized with phenyl isothiocyanate and 4-sulfophenyl isothiocyanate.

Photodissociation (PD) tandem mass spectra have been obtained at 266 nm for the protonated molecules of a tryptic peptide, ASHLGLAR, and of its phenyl isothiocyanate and 4-sulfophenyl isothiocyanate derivatives, generated by matrix-assisted laser desorption/ionization. Derivatization with the aromatic chromophores greatly reduced the intensity of the laser required for efficient PD. Major fragment ions observed in the three spectra are quite similar. General features of the PD tandem mass spectra and their potential utility for analytical purposes are discussed.