Relative Ion Intensities Research Articles

Optimization of a GC/MS procedure that uses parallel factor analysis for the determination of bisphenols and their diglycidyl ethers after migration from polycarbonate tableware

Bisphenol A (BPA), bisphenol F (BPF) and their corresponding diglycidyl ethers (BADGE and BFDGE) are simultaneously determined using a programmed-temperature vaporizer-gas chromatography/mass spectrometry (PTV-GC/MS) system. BPA is used in the production of polycarbonate (PC), whereas BADGE and BFDGE are for manufacturing epoxy resins. Several food alerts caused by the migration of this kind of substances from contact food materials have led to the harmonization of the European legislation in Commission Regulation (EU) No. 10/2011, in force from 14 January 2011. In consequence, the use of BPA has been prohibited in the manufacture of plastic infant feeding bottles from 1 May 2011 and from 1 June 2011 regarding the placing on the market and importation into the European Union. Recently, the French Parliament has decreed that the presence of BPA in any food containers will be banned. Similarly, the use and/or presence of BFDGE are not allowed.In this work, a GC/MS method has been developed for the simultaneous determination of BPF, BPA, BFDGE and BADGE. For each one of the I samples that are analyzed, the abundance of J characteristic m/z ratios is recorded at K times around the retention time of each peak, so a data tensor of dimension I×J×K is obtained for every analyte. The decomposition of this tensor by means of parallel factor analysis (PARAFAC) enables to: (a) identify unequivocally each analyte according to the maximum permitted tolerances for relative ion intensities, and (b) quantify each analyte, even in the presence of coeluents. This identification, based on the mass spectrum and the retention time, guarantees the specificity of the analysis. This specificity could fail if the total ion chromatogram (TIC) is considered when there is poor resolution between some peaks or whether interferents coelute.With the aim of studying the effect of shortening the time of the analysis on the quality of the determinations while maintaining the specificity of the identifications, two of the heating ramps in the oven temperature program are changed according to a two-level factorial design. Each analyte is identified by means of a PARAFAC decomposition of a data tensor obtained from several concentration levels, in such a way that five figures of merit are calculated for each experiment of the design. The analysis of these figures of merit for the 16 objects (4 compounds×4 heating ramps) using principal component analysis (PCA) shows that the shortest temperature program should be considered, since this is the one the best figures of merit for BPA and BFDGE (both banned) are achieved with. At these conditions and with probabilities of false positive and false negative fixed at 0.05, values of detection capability (CCβ) between 2.65 and 4.71μgL−1 when acetonitrile is the injection solvent, and between 1.97 and 5.53μgL−1 when acetone, are obtained.This GC/MS method has been applied to the simultaneous determination of BPF, BPA, BFDGE and BADGE in food simulant D1 (ethanol–H2O, 1:1 v/v), which had been previously in contact with PC tableware for 24h at 70°C and then pretreated by a solid-phase extraction (SPE) step. The migration of BPA from the new PC containers analyzed is confirmed, and values between 104.67 and 181.46μgL−1 (0.73 and 1.27μgL−1 after correction) of BPA have been estimated. None of the results obtained exceeds the specific migration limit of 600μgL−1 established by law for BPA in plastic food materials different from PC infant feeding bottles. Severe problems of coelution of interferents have been overcome using PARAFAC decompositions in the analysis of these food simulant samples.

A novel rapid detection approach for the analysis of radionuclides in environmental samples using graphite MALDI mass spectrometry

Initial studies into the analysis of Uranium and Lanthanide salts by graphite matrix assisted laser desorption ionisation–time of flight–mass spectrometry (GALDI–ToF–MS) are presented. It is shown that GALDI–ToF–MS represents a more effective method for ion generation compared to that observed by direct laser irradiation, providing more sensitive analysis and enhanced mass spectral quality. Assessment of sensitivity, detection limits and isotope signature determination is given with absolute detection limits having been found to be in the low ng to pg range and abundance measurements being consistent with expected values to within 2–10 % depending on the relative isotope ion intensities. Using imaging software, GALDI–ToF–MS can be used to identify the presence of low micron-sized uranium oxide particles and establish their distribution across a substrate surface to enable particle location and in so doing identify particles of interest which are of non-natural, enriched content. In this context, GALDI–ToF–MS is proposed as a novel screening method for uranium containing micro-particles which has potential as a rapid and low cost means for particle location to enable effective sampling using micro-manipulation and optical microscopy, for onward isotope ratio mass spectrometry.

Calculations of relative intensities of fragment ions in the MSMS spectra of a doubly charged penta-peptide

BackgroundCurrently, the tandem mass spectrometry (MSMS) of peptides is a dominant technique used to identify peptides and consequently proteins. The peptide fragmentation inside the mass analyzer typically offers a spectrum containing several different groups of ions. The mass to charge (m/z) values of these ions can be exactly calculated following simple rules based on the possible peptide fragmentation reactions. But the (relative) intensities of the particular ions cannot be simply predicted from the amino-acid sequence of the peptide. This study presents initial work towards developing a theoretical fundamental approach to ion intensity elucidation by utilizing quantum mechanical computations.MethodsMSMS spectra of the doubly charged GAVLK peptide were collected on electrospray ion trap mass spectrometers using low energy modes of fragmentation. Density functional theory (DFT) calculations were performed on the population of ion precursors to determine the fragment ion intensities corresponding to a Boltzmann distribution of the protonation of nitrogens in the peptide backbone amide bonds.ResultsWe were able to a) predict the y and b ions intensities order in concert with the experimental observation; b) predict relative intensities of y ions with errors not exceeding the experimental variation.ConclusionsThese results suggest that the GAVLK peptide fragmentation process in the ion trap mass spectrometer is predominantly driven by the thermodynamic stability of the precursor ions formed upon ionization of the sample. The computational approach presented in this manuscript successfully calculated ion intensities in the mass spectra of this doubly charged tryptic peptide, based solely on its amino acid sequence. As such, this work indicates a potential of incorporating quantum mechanical calculations into mass spectrometry based algorithms for molecular identification.

Increasing the Multiplexing Capacity of TMTs Using Reporter Ion Isotopologues with Isobaric Masses

Quantitative mass spectrometry methods offer near-comprehensive proteome coverage; however, these methods still suffer with regards to sample throughput. Multiplex quantitation via isobaric chemical tags (e.g., TMT and iTRAQ) provides an avenue for mass spectrometry-based proteome quantitation experiments to move away from simple binary comparisons and toward greater parallelization. Herein, we demonstrate a straightforward method for immediately expanding the throughput of the TMT isobaric reagents from 6-plex to 8-plex. This method is based upon our ability to resolve the isotopic shift that results from substituting a (15)N for a (13)C. In an accommodation to the preferred fragmentation pathways of ETD, the TMT-127 and -129 reagents were recently modified such that a (13)C was exchanged for a (15)N. As a result of this substitution, the new TMT reporter ions are 6.32 mDa lighter. Even though the mass difference between these reporter ion isotopologues is incredibly small, modern high-resolution and mass accuracy analyzers can resolve these ions. On the basis of our ability to resolve and accurately measure the relative intensity of these isobaric reporter ions, we demonstrate that we are able to quantify across eight samples simultaneously by combining the (13)C- and (15)N-containing reporter ions. Considering the structure of the TMT reporter ion, we believe this work serves as a blueprint for expanding the multiplexing capacity of the TMT reagents to at least 10-plex and possibly up to 18-plex.

Liquid chromatography tandem mass spectrometry for the simultaneous determination of mequindox and its metabolites in porcine tissues

A rapid liquid chromatography tandem mass spectrometric method was developed for the simultaneous determination of mequindox and its five metabolites (2-isoethanol mequindox, 2-isoethanol 1-desoxymequindox, 1-desoxymequindox, 1,4-bisdesoxymequindox, and 2-isoethanol bisdesoxymequindox) in porcine muscle, liver, and kidney, fulfilling confirmation criteria with two transitions for each compound with acceptable relative ion intensities. The method involved acid hydrolysis, purification by solid-phase extraction, and subsequent analysis with liquid chromatography tandem mass spectrometry using electrospray ionization operated in positive polarity with a total run time of 15 min. The decision limit values of five analytes in porcine tissues ranged from 0.6 to 2.9 μg/kg, and the detection capability values ranged from 1.2 to 5.7 μg/kg. The results of the inter-day study, which was performed by fortifying porcine muscle (2, 4, and 8 μg/kg), liver, and kidney (10, 20, and 40 μg/kg) samples on three separate days, showed that the accuracy of the method for the various analytes ranged between 75.3 and 107.2% with relative standard deviation less than 12% for each analyte.

Fragmentation of oxime and silyl oxime ether odd‐electron positive ions by the McLafferty rearrangement: new insights on structural factors that promote α,β fragmentation

The McLafferty rearrangement is an extensively studied fragmentation reaction for the odd-electron positive ions from a diverse range of functional groups and molecules. Here, we present experimental and theoretical results of 12 model compounds that were synthesized and investigated by GC-TOF MS and density functional theory calculations. These compounds consisted of three main groups: carbonyls, oximes and silyl oxime ethers. In all electron ionization mass spectra, the fragment ions that could be attributed to the occurrence of a McLafferty rearrangement were observed. For t-butyldimethylsilyl oxime ethers with oxygen in a β-position, the McLafferty rearrangement was accompanied by loss of the t-butyl radical. The various mass spectra showed that the McLafferty rearrangement is relatively enhanced compared with other primary fragmentation reactions by the following factors: oxime versus carbonyl, oxygen versus methylene at the β-position and ketone versus aldehyde. Calculations predict that the stepwise mechanism is favored over the concerted mechanism for all but one compound. For carbonyl compounds, C-C bond breaking was the rate-determining step. However, for both the oximes and t-butyldimethylsilyl oxime ethers with oxygen at the β-position, the hydrogen transfer step was rate limiting, whereas with a CH(2) group at the β-position, the C-C bond breaking was again rate determining. n-Propoxy-acetaldehyde, bearing an oxygen atom at the β-position, is the only case that was predicted to proceed through a concerted mechanism. The synthesized oximes exist as both the (E)- and (Z)-isomers, and these were separable by GC. In the mass spectra of the two isomers, fragment ions that were generated by the McLafferty rearrangement were observed. Finally, fragment ions corresponding to the McLafferty reverse charge rearrangement were observed for all compounds at varying relative ion intensities compared with the conventional McLafferty rearrangement.

Simultaneous Determination of Xylazine, Free Morphine, Codeine, 6-Acetylmorphine, Cocaine and Benzoylecgonine in Postmortem Blood by UPLC-MS-MS

Xylazine, a veterinary sedative, has been found as an adulterant of heroin in street drugs in Puerto Rico. It was found in combination with free morphine and 6-acetylmorphine, codeine, cocaine and benzoylecgonine in postmortem cases at the Puerto Rico Institute of Forensic Sciences (PRIFS). Xylazine is not approved for human use because it has been proven harmful. Currently, three separate analyses are required to determine all the aforementioned drugs at the PRIFS's toxicology laboratory. To reduce analysis time consumption, sample volume, run time, sample preparation and cost, a high-throughput ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of xylazine, free morphine, 6-acetylmorphine, codeine, cocaine and benzoylecgonine in 0.25 mL postmortem blood by protein precipitation, fulfilling confirmation criteria with three transitions for each compound with acceptable relative ion intensities. Linearity was established between 10-1,000 ng/mL. Total run time was 2.5 min. Limit of detection was 1 ng/mL for cocaine and xylazine, 2 ng/mL for 6-acetylmorphine and 10 ng/mL for free morphine, codeine and benzoylecgonine. The intra-day and inter-day precision and accuracy was less than 15.6%. Process efficiencies ranged from 35.9 to 123.4% and recoveries from 59.9 to 110.1%. The developed method was successfully applied to casework.

Determination of Perfluorochemicals in Human Milk Using Isotope‐dilution Liquid Chromatography Tandem Mass Spectrometry

AbstractA highly precise and accurate analytical method utilizing an isotope‐dilution liquid chromatography tandem mass spectrometry was developed and validated to determine two perfluorochemicals (PFCs): perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) in human milk samples. Identification of the analytes was confirmed under negative electrospray with multiple reaction monitoring (MRM) mode by the monitoring of one precursor ion and two product ions, and matching of relative ion intensities of the ions concerned in samples and calibration standards. Quantitation was based on the measurement of concentration ratios of the natural and labeled‐analogues in the samples and calibration mixtures. The isotope‐labeled internal standards were also used to correct the matrix effect and variations associated with the analysis. Intra‐ and inter‐day repeatabilities of replicate analyses of the PFOA and PFOS in milk samples were below 8%. The limit of quantitation was 2 pg/mL in a 5 mL milk sample. The PFOA and PFOS were detected in all 20 human milk samples at concentrations from 27.0 to 207 pg/mL. This is the first study to measure the occurrence of PFOA and PFOS in human milk from Taiwan.

Collision induced dissociation study of the major components of silymarin

The fragmentation properties of the major silymarin constituents, including silydianin, silychristin A and B, silybin A and B and isosilybin A and B were studied by energy-dependent collision-induced dissociation (CID) technique. The survival yield (SY) method was applied to compare the fragmentation behavior of the various silymarin components. MS/MS spectra were recorded at collision energies which correspond to about 50% survival yield (CE50) and about 1% survival yield (CE01). The laboratory frame collision energy dependence of the fragmentation was studied on the basis of the breakdown curves, i.e., the relative intensities of fragment ions versus collision energy. The fragmentation of silybins and isosilybins is distinct in spite of the minimal difference between their structures. This phenomenon, which is the basis of their analytical identification, is explained in details for the first time, on the basis of the energy-dependent MS/MS experiments and the conformational analysis.

An improved gas chromatography screening method for doping substances using triple quadrupole mass spectrometry, with an emphasis on quality assurance

A GC-QqQ-MS method was developed for the detection of over 150 compounds from different classes (steroids, narcotics, stimulants, β-blockers, β-2-agonists and hormone antagonists) in a qualitative way. In the quantitative part, the traditional steroid profile with the most important endogenous steroids is expanded with six minor metabolites, which further improves the detection and identification of endogenous steroid abuse. In addition to these, norandrosterone, salbutamol and the major metabolite of cannabis are also quantified. Methods developed for anti-doping purposes should be subjected to the highest level of quality. Here, the addition of a combination of (deuterated) internal standards allows for an accurate quality control of every single step of the methodology: hydrolysis efficiency, derivatization efficiency and microbiological degradation are monitored in every single sample. Additionally, special attention is paid to the relationships between parameters indicating degradation by micro-organisms and the reliability of the steroid profile. The impact of the degradation is studied by evaluation of the quantities and percentages of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione. The concept of measurement uncertainty was introduced for the evaluation of relative abundances of mass-to-charge ratios and the obtained ranges were compared with the World Anti-Doping Agency regulations on tolerance windows for relative ion intensities. The results indicate that the approaches are similar.

Investigation of peptide size, residue position, neighbor amino acid and side chain effect on macrocyclization of bn (n = 5–7) ions

A systematic study was carried out to examine the effects of the side chain, peptide size, residue position, and neighboring amino acid on the macrocyclization of b ions. The work utilized isomeric model peptides YAGFLV-NH2, AGFLVY-NH2, GFLVYA-NH2, FLVYAG-NH2, LVYAGF-NH2, VYAGFL-NH2, which all have the same amino acid sequence in cyclic form. The b6 ions derived from all these isomeric peptides form the same macrocyclic structure due to the generation of the same amino acid sequence order upon cyclization. Hence, the MS/MS spectra and breakdown graphs of b6 ions derived from these peptides are similar to each other. However, the relative intensities of the non-direct sequence ions in both the MS/MS spectra and breakdown graphs of the b6 ions derived from FAYVGL-NH2, GVYALF-NH2 and VFYLAG-NH2 show a different distribution from each other and the first series, even though they are all isomeric peptides. This could be due to the different amino acid sequence order in the cyclic forms of these peptides. It is clearly shown that the neighboring amino acid influences the selective opening of the macrocyclic form.Additionally, XYAGFLV-NH2 and YAGXFLV-NH2 (where X=C, D, E, H, K, M, N, P, Q, S, T, and W are amino acid residues) were also studied in order to examine the influence of the peptide size, amino acid side chain, and position on the ring formation and cleavage of macrocyclic b5, b6 and b7 ions. The results have clearly shown that b6 and b7 ions have a higher tendency of macrocyclization compared to b5 ions with the exception of QYAGFLV-NH2. Additionally, it was observed that selective ring opening is also dependent on the size of the b ions and the position of the amino acid residue. From our study of the macrocyclic b6 ions of our model peptides, the Q, W, K, and M residues were found to be more favorable eliminations when compared to C, D, E, H, N, P, S, and T. Based on the results, no preferential cleavage order can be specified depending on the nature of amino acid side chain.

The influence of ionization efficiency on the multiply charged ions produced by laser-clusters interaction

The productions of multiply charged ions in the interactions of intense nanosecond laser pulse with clusters have aroused broad interests in molecular physics. Benzene, cyclohexene and cyclohexane clusters are chosen to study the effect of multiphoton ionization (MPI) efficiency on the relative intensity of multiply charged ions, as they possess similar molecular structures and the same element constitutions. They are ionized with a 5 ns Nd-YAG nanosecond laser. The carbon charge state produced by cyclohexene and cyclohexane is about 4; while by benzene is only about 3. The ratios of C3+/C2+ for cyclohexane, cyclohexene and benzene are 1.1, 0.6 and 0.4, respectively. The relative MPI efficiencies of three molecules are measured to be in the magnitude sequence of benzene > cyclohexene > cyclohenane by diffusion beam. Higher MPI efficiency of molecules can cause more than one molecules to be ionized at the edge of laser pulse, the Coulomb repelling force between adjacent ions leads clusters to early split into small size ones, which will prevent the production of the highly charged ions.

High-throughput mass finger printing and Lewis blood group assignment of human milk oligosaccharides

The structural diversity of human milk oligosaccharides (HMOs) strongly depends on the Lewis (Le) blood group status of the donor which allows a classification of these glycans into three different groups. Starting from 50 μL of human milk, a new high-throughput, standardized, and widely automated mass spectrometric approach has been established which can be used for correlation of HMO structures with the respective Lewis blood groups on the basis of mass profiles of the entire mixture of glycans together with selected fragment ion spectra. For this purpose, the relative abundance of diagnostically relevant compositional species, such as Hex(2)Fuc(2) and Hex(3)HexNAc(1)Fuc(2), as well as the relative intensities of characteristic fragment ions obtained thereof are of key importance. For each Lewis blood group, i.e., Le(a-b+), Le(a+b-), and Le(a-b-), specific mass profile and fragment ion patterns could be thus verified. The described statistically proven classification of the derived glycan patterns may be a valuable tool for analysis and comparison of large sets of milk samples in metabolic studies. Furthermore, the outlined protocol may be used for rapid screening in clinical studies and quality control of milk samples donated to milk banks.

Increased Selectivity, Analytical Precision, and Throughput in Targeted Proteomics

Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.

Proteomic Analysis of Native Hepatocyte Nuclear Factor-4α (HNF4α) Isoforms, Phosphorylation Status, and Interactive Cofactors

Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor that has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady-state remains to be elucidated. Here we report the native HNF4α isoform, phosphorylation status, and complexes in the steady-state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semiquantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and hepatocyte nuclear factor-4γ was found. A biochemical and genomewide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here are important for an accurate understanding of transcriptional regulation.

Analysis of personal care products on model skin surfaces using DESI and PADI ambient mass spectrometry

Two ambient ionisation techniques, desorption electrospray ionisation (DESI) and plasma assisted desorption ionisation (PADI), have been used to analyse personal care products (PCPs) on fixed fibroblast cell surfaces. The similarities and differences between the two techniques for this type of analysis have been explored in various ways. Here, we show the results of DESI and PADI analysis of individual PCP ingredients as well as the analysis of these as complex creams on model skin surfaces, with minimal sample preparation. Typically, organosiloxanes and small molecules were detected from the creams. A study of the morphological damage of the fibroblast cells by the two ionisation techniques showed that for a less than 10% reduction in cell number, acquisition times should be limited to 5 s for PADI, which gives good signal levels; with DESI, the morphological damage was negligible. The operating parameters for the plasma source were optimised, and it was also found that the parameters could be modified to vary the relative intensity of different ions in the mass spectrum.

Rapid analysis of fructans and comparison of fructan profiles in several different types of asparagus storage roots using MALDI-TOF MS

SummaryAppropriate conditions for analysing fructans in the storage roots of field-grown asparagus using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) were investigated. High resolution and ionisation were achieved when lyophilised and ground storage root powders were extracted twice with 1.0 ml distilled and deionised hot water (90°C), supplemented with 1.0 ml of 100% (v/v) ethanol, concentrated to approx. 1.0 ml, and mixed with 10 g l–1 2,5-dihydroxybenzoic acid (DHB) at 1:1 (v/v) before loading on a sample slide for MALDI-TOF MS, which was performed in the linear positive ion mode at a laser intensity of 15 kV. The mass spectral profiles showed that the degree of polymerisation (DP) of fructans ranged from three-to-21 in storage roots collected in the Winter.When different amounts of -cyclodextrin were added to each sample, a linear positive relationship was observed between the amount of -cyclodextrin and the relative positive ion intensity. With -cyclodextrin as an internal standard, fructan profiles were compared between Summer- and Winter-collected asparagus storage root samples. Furthermore, the fructan profiles of three different types of storage root of different colours (i.e., milky-white, yellow, or dark-brown) were also examined. Fructan contents were always higher in the Winter-collected samples than in those collected in the Summer, regardless of the DP. However, samples from the different coloured roots showed variable fructan profiles, depending on the DP. These results indicate that fructan analysis using MALDI-TOF MS would be useful for examining the dynamics of carbohydrate metabolism in asparagus storage roots because of its high degree of accuracy and its simplicity.

VAMAS interlaboratory study on organic depth profiling. Part I: Preliminary report

AbstractWe present preliminary results of a VAMAS interlaboratory study on organic depth profiling, TWA2, sub‐project A3 (d). A layered organic system was used to assess the repeatability and comparability of organic depth profiling. Nineteen respondents have provided sufficient data to demonstrate that the coefficients of variation for depth resolution, sputtering yield and relative secondary ion intensities are typically 5–10%. This can be as good as 2–3% with modern instruments, when using a stable sputtering ion current and careful procedures, which approaches the limits set by the samples themselves. The respondents have also demonstrated three methods to improve the quality of depth profiling for this system, namely, sample rotation, cooling to at least − 80 °C and grazing incidence angles for the sputtering ion. Copyright © 2010 John Wiley & Sons, Ltd.

IsobariQ: Software for Isobaric Quantitative Proteomics using IPTL, iTRAQ, and TMT

Isobaric peptide labeling plays an important role in relative quantitative comparisons of proteomes. Isobaric labeling techniques utilize MS/MS spectra for relative quantification, which can be either based on the relative intensities of reporter ions in the low mass region (iTRAQ and TMT) or on the relative intensities of quantification signatures throughout the spectrum due to isobaric peptide termini labeling (IPTL). Due to the increased quantitative information found in MS/MS fragment spectra generated by the recently developed IPTL approach, new software was required to extract the quantitative information. IsobariQ was specifically developed for this purpose; however, support for the reporter ion techniques iTRAQ and TMT is also included. In addition, to address recently emphasized issues about heterogeneity of variance in proteomics data sets, IsobariQ employs the statistical software package R and variance stabilizing normalization (VSN) algorithms available therein. Finally, the functionality of IsobariQ is validated with data sets of experiments using 6-plex TMT and IPTL. Notably, protein substrates resulting from cleavage by proteases can be identified as shown for caspase targets in apoptosis.

VAMAS interlaboratory study on organic depth profiling

AbstractWe present the results of a VAMAS (Versailles project on Advanced Materials and Standards) interlaboratory study on organic depth profiling, in which twenty laboratories submitted data from a multilayer organic reference material. Individual layers were identified using a range of different sputtering species (C60n+, Cs+, SF5+ and Xe+), but in this study only the C60n+ ions were able to provide truly ‘molecular’ depth profiles from the reference samples. The repeatability of profiles carried out on three separate days by participants was shown to be excellent, with a number of laboratories obtaining better than 5% RSD (relative standard deviation) in depth resolution and sputtering yield, and better than 10% RSD in relative secondary ion intensities. Comparability between laboratories was also good in terms of depth resolution and sputtering yield, allowing useful relationships to be found between ion energy, sputtering yield and depth resolution. The study has shown that organic depth profiling results can, with care, be compared on a day‐to‐day basis and between laboratories. The study has also validated three approaches that significantly improve the quality of organic depth profiling: sample cooling, sample rotation and grazing angles of ion incidence. © Crown copyright 2010.