Quadrupole Isolation Research Articles

High-resolution quadrupole improves spectral purity and reduces interference from non-target ions in isobaric multiplexed quantitative proteomics

BackgroundMass spectrometry (MS)-based proteomics is a powerful tool for identifying and quantifying proteins. However, chimeric spectra caused by the fragmentation of multiple precursors within the same isolation window impair the accuracy of peptide identification and isobaric mass tag-based quantification. While there have been advances in computational deconvolution of chimeric spectra and methods to further separate the peptides by ion mobility or through MSn, the use of narrower isolation windows to decrease the fraction of chimeric species remains to be fully explored. ResultsWe present results obtained on a SCIEX TripleTOF instrument where the quadrupole was optimized and tuned for precursor isolation at 0.1 Da (FWHH). Using a three-proteome model (trypsin digest of protein lysates from yeast, human and E. coli) and 8-plex iTRAQ labeling to document the interference effect, we investigated the impact of co-fragmentation on spectral purity, identification accuracy and quantification accuracy. The narrow quadrupole isolation window significantly improved the spectral purity and reduced the interference of non-target precursors on quantification accuracy. The high-resolution isolation strategy also reduced the number of false identifications caused by chimeric spectra. While these improvements came at the cost of sensitivity loss, combining high-resolution isolation with other advanced techniques, including ion mobility, may result in improved accuracy in identification and quantification. SignificanceCompared to standard-resolution quadrupole isolation (0.7 Da), high-resolution quadrupole isolation (0.1 Da) significantly improved the spectral purity and quantification accuracy while reducing the number of potential false identifications caused by chimeric spectra, thus showing excellent potential for further development to analyze clinical proteomics samples, for which high accuracy is essential.

The use of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a data-independent acquisition high-resolution mass spectrometry approach, in forensic toxicological regimes: A review.

Sequential window acquisition of all theoretical fragment ion spectra (SWATH) is a type of high-resolution mass spectrometry that uses data-independent acquisition. Compared with more targeted acquisition schemes, the power behind this data-independent acquisition technique comes from its ability to mitigate interferences via the use of SWATH acquisition windows (Q1 quadrupole isolation windows) while still obtaining all accurate mass information. However, consistent with high-resolution mass spectrometry techniques, its routine and high throughput implementation in forensic toxicology is limited due to the complex processing power required to effectively manage the large amount of acquired data. It is therefore pivotal to create an efficient and validated identification criterion that confidently reports suspected positive detections as a confirmational technique for final reporting. This review examines all publications that implemented SWATH in a forensic toxicological framework with suggestive best practices and commonly used criteria. Seventeen publications were reviewed for extraction, liquid chromatography and mass spectrometry parameters, and more specifically for all SWATH applicable characteristics including spray voltages, collision energies and spreads, mass error, isotopic ratio difference, retention time error, and library score thresholds. Notwithstanding the challenges SWATH implementation faces for a laboratory, the technique demonstrates its potential to be utilized in routine forensic toxicology testing regimes and aids in the detection of both common and emerging novel drugs simultaneously.

Two-Dimensional Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of N-Linked Glycopeptides.

Glycosylation is a common modification across living organisms and plays a central role in understanding biological systems and disease. Our ability to probe the gylcome has grown exponentially in the past several decades. However, further improvements to the analytical toolbox available to researchers would allow for increased capabilities to probe structure and function of biological systems and to improve disease treatment. This article applies the developing technique of two-dimensional Fourier transform ion cyclotron resonance mass spectrometry to a glycoproteomic workflow for the standard glycoproteins coral tree lectin (CTL) and bovine ribonuclease B (BRB) to demonstrate its feasibility as a tool for glycoproteomic workflows. 2D infrared multiphoton dissociation and electron capture dissociation spectra of CTL reveal comparable structural information to their 1D counterparts, confirming the site of glycosylation and monosaccharide composition of the glycan. Spectra collected in 2D of BRB reveal correlation lines of fragment ion scans and vertical precursor ion scans for data collected using infrared multiphoton dissociation and diagonal cleavage lines for data collected by electron capture dissociation. The use of similar techniques for glycoproteomic analysis may prove valuable in instances where chromatographic separation is undesirable or quadrupole isolation is insufficient.

Digital Quadrupole Isolation and Electron Capture Dissociation on an Extended Mass Range Q-TOF Provides Sequence and Structure Information on Proteins and Protein Complexes.

Electron capture dissociation (ECD) is now a well-established method for sequencing peptides and performing top-down analysis on proteins of less than 30 kDa, and there is growing interest in using this approach for studies of larger proteins and protein complexes. Although much progress on ECD has been made over the past few decades, establishing methods for obtaining informative spectra still poses a significant challenge. Here we describe how digital quadrupole (DigiQ) ion isolation can be used for the mass selection of single charge states of proteins and protein complexes prior to undergoing ECD and/or charge reduction. First, we demonstrate that the DigiQ can isolate single charge states of monomeric proteins such as ubiquitin (8.6 kDa) and charge states of large protein complexes such as pyruvate kinase (234 kDa) using a hybrid quadrupole-TOF-MS (Agilent extended m/z range 6545XT). Next, we demonstrate that fragment ions resulting from ECD can be utilized to provide information about the sequence and structure of the cytochrome c/heme complex and the ubiquitin monomer. Lastly, an especially interesting result for DigiQ isolation and electron capture (EC) was noted; namely, the 16+ charge state of the streptavidin/biotin complex reveals different electron capture patterns for the biotinylated proteoforms of streptavidin. This result is consistent with previous reports that apo streptavidin exists in multiple conformations and that biotin binding shifts the conformational dynamics of the complex (Quintyn, R. Chem. Biol. 2015, 22 (55), 583-592).

New Insights into the Intrinsic Electron-Based Dissociation Behavior of Cytochrome c Oligomers.

Between 2003 and 2017, four reports were published that demonstrated the intrinsic ability of the native iron-containing proteins cytochrome c and ferritin to undergo radical-based backbone fragmentation in the gas phase without the introduction of exogenous electrons. For cytochrome c in particular, this effect has so far only been reported to occur in the ion source, preventing the in-depth study of reactions occurring after gas-phase isolation of specific precursors. Here, we report the first observation of this intrinsic native electron capture dissociation behavior after quadrupole isolation of specific charge states of the cytochrome c dimer and trimer, providing direct experimental support for key aspects of the mechanism proposed 20 years ago. Furthermore, we provide evidence that, in contrast to some earlier proposals, these oligomeric states are formed in bulk solution rather than during the electrospray ionization process and that the observed fragmentation site preferences can be rationalized through the structure and interactions within these native oligomers rather than the monomer. We also show that the observed fragmentation pattern─and indeed, whether or not fragmentation occurs─is highly sensitive to the provenance and history of the protein samples, to the extent that samples can show distinct fragmentation behavior despite behaving identically in ion mobility experiments. This rather underexplored method therefore represents an exquisitely sensitive conformational probe and will hopefully receive more attention from the biomolecular mass spectrometry community in the future.

Resolving isobaric interferences in direct infusion tandem mass spectrometry.

RationaleThe co‐fragmentation of precursors in direct infusion (DI) tandem high‐resolution mass spectrometry (HRMS) can complicate the fragment spectra and consequently lead to false hits during compound identification.MethodsThe method herein described, termed IQAROS (incremental quadrupole acquisition to resolve overlapping spectra), modulates the intensities of precursors and fragments by stepwise movement of the quadrupole isolation window over the mass‐to‐charge (m/z) range of the precursors. The modulated signals are then deconvoluted by a linear regression model to reconstruct the fragment spectra with less interference. The hardware to demonstrate the use of IQAROS was an orbitrap with electrospray ionization (ESI) or secondary electrospray ionization (SESI), although the method can also be applied to other ionization techniques or mass analyzers.ResultsAssessing the performance of IQAROS with isobaric standards revealed that the reconstructed fragment spectra match with spectra acquired from the pure standards and that more compounds were correctly identified compared with the classical approach with the quadrupole centered at the m/z value of the precursor of interest. Moreover, the strength of IQAROS is exemplified by the identification of two isobaric biomarkers directly from a breath sample with SESI‐HRMS.ConclusionsWith IQAROS, cleaner fragment spectra of co‐fragmenting isobars during DI‐HRMS analysis can be obtained. IQAROS can easily be set up by the standard graphical user interface of the instrument. Therefore, it facilitates the characterization of features of interest in samples analyzed by DI‐HRMS, for example, in high‐throughput or real‐time metabolomics.

Data-Independent Identification of Suspected Organic Pollutants Using Gas Chromatography-Atmospheric Pressure Chemical Ionization-Mass Spectrometry.

The identity of an unknown environmental pollutant is reflected by the mass and dissociation chemistry of its (quasi)molecular ion. Gas chromatography-atmospheric pressure chemical ionization-mass spectrometry (GC-APCI-MS) increases the yield of molecular ions (compared to conventional electron ionization) by collisional cooling. Scanning quadrupole data-independent acquisition (SQDIA) permits unbiased, unattended selection of (quasi)molecular ions and acquisition of structure-diagnostic collision-induced dissociation mass spectra, while minimizing interferences, by sequentially cycling a quadrupole isolation window through the m/z range. This study reports on the development of a suspect screening method based on industrial compounds with bioaccumulation potential. A comparison of false and correct identifications in a mixed standard containing 30 analytes suggests that SQDIA results in a markedly lower false-positive rate than standard DIA: 5 for SQDIA and 82 for DIA. Electronic waste dust was analyzed using GC and quadrupole time-of-flight MS with APCI and SQDIA acquisition. A total of 52 brominated, chlorinated, and organophosphorus compounds were identified by suspect screening; 15 unique elemental compositions were identified using nontargeted screening; 17 compounds were confirmed using standards and others identified to confidence levels 2, 3, or 4. SQDIA reduced false-positive identifications, compared to experiments without quadrupole isolation. False positives also varied by class: 20% for Br, 37% for Cl, 75% for P, and >99% for all other classes. The structure proposal of a previously reported halogenated compound was revisited. The results underline the utility of GC-SQDIA experiments that provide information on both the (quasi)molecular ions and its dissociation products for a more confident structural assignment.

Structural Characterization of Dissolved Organic Matter at the Chemical Formula Level Using TIMS-FT-ICR MS/MS.

TIMS-FT-ICR MS is an important alternative to study the isomeric diversity and elemental composition of complex mixtures. While the chemical structure of many compounds in the dissolved organic matter (DOM) remains largely unknown, the high structural diversity has been described at the molecular level using chemical formulas. In this study, we further push the boundaries of TIMS-FT-ICR MS by performing chemical formula-based ion mobility and tandem MS analysis for the structural characterization of DOM. The workflow described is capable to mobility select (R ∼ 100) and isolate molecular ion signals (Δm/z = 0.036) in the ICR cell, using single-shot ejections after broadband ejections and MS/MS based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID). The workflow results are compared to alternative TIMS-q-FT-ICR MS/MS experiments with quadrupole isolation at nominal mass (∼1 Da). The technology is demonstrated with isomeric and isobaric mixtures (e.g., 4-methoxy-1-naphthoic acid, 2-methoxy-1-naphthoic acid, decanedioic acid) and applied to the characterization of DOM. The application of this new methodology to the analysis of a DOM is illustrated by the isolation of the molecular ion [C18H18O10-H]- in the presence of other isobars at nominal mass 393. Five IMS bands were assigned to the heterogeneous ion mobility profile of [C18H18O10-H]-, and candidate structures from the PubChem database were screened based on their ion mobility and the MS/MS matching score. This approach overcomes traditional challenges associated with the similarity of fragmentation patterns of DOM samples (e.g., common neutral losses of H2O, CO2, and CH2-H2O) by narrowing down the isomeric candidate structures using the mobility domain.

Advantages of Two-Dimensional Electron-Induced Dissociation and Infrared Multiphoton Dissociation Mass Spectrometry for the Analysis of Agrochemicals.

Analysis of agrochemicals in an environmental matrix is challenging because these samples contain multiple agrochemicals, their metabolites, degradation products, and endogenous compounds. The analysis of such complex samples is achieved using chromatographic separation techniques coupled to mass spectrometry. Herein, we demonstrate a two-dimensional mass spectrometry (2DMS) technique on a 12 T Fourier transform ion cyclotron resonance mass spectrometer that can analyze a mixture of agrochemicals without using chromatography or quadrupole isolation in a single experiment. The resulting 2DMS contour plot contains abundant tandem MS information for each component in the sample and correlates product ions to their corresponding precursor ions. Two different fragmentation methods are employed, infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID), with 2DMS to analyze the mixture of singly charged agrochemicals. The product ions of one of the agrochemicals, pirimiphos-methyl, present in the sample was used to internally calibrate the entire 2DMS spectrum, achieving sub part per million (ppm) to part per billion (ppb) mass accuracies for all species analyzed. The work described in this study will show the advantages of the 2DMS approach, by grouping species with common fragments/core structure and mutual functional groups, using precursor lines and neutral loss lines. In addition, the rich spectral information obtained from IRMPD and EID 2DMS contour plots can accurately identify and characterize agrochemicals.

Extending the Separation Space with Trapped Ion Mobility Spectrometry Improves the Accuracy of Isobaric Tag-Based Quantitation in Proteomic LC/MS/MS.

Two-dimensional separation by nano-LC and trapped ion mobility spectrometry (TIMS) prior to Q/TOF tandem mass spectrometry significantly improves the accuracy of isobaric tag-based quantitation in proteome analysis without the need for additional measurement time for TIMS insertion between LC and Q/TOF MS. The obtained peak capacity of up to 3300 h-1 in LC/TIMS reduced the coisolation of precursor ions at the quadrupole analyzer, resulting in more accurate ratios of reporter ions derived from isobaric tags in product ion spectra obtained at the TOF analyzer. We also found that TIMS with a narrower quadrupole isolation window could reduce the ratio compression effect at least as effectively as the synchronous precursor selection method using MS3 scans without compromising sensitivity or coverage. Our results suggest that the 65 min gradient LC/TIMS/Q/TOF system is an excellent platform for high-throughput proteomics studies.

LESA Cyclic Ion Mobility Mass Spectrometry of Intact Proteins from Thin Tissue Sections.

Liquid extraction surface analysis (LESA) is an ambient surface sampling technique that allows the analysis of intact proteins directly from tissue samples via mass spectrometry. Integration of ion mobility separation to LESA mass spectrometry workflows has shown significant improvements in the signal-to-noise ratios of the resulting protein mass spectra and hence the number of proteins detected. Here, we report the use of a quadrupole–cyclic ion mobility–time-of-flight mass spectrometer (Q-cIM-ToF) for the analysis of proteins from mouse brain and rat kidney tissues sampled via LESA. Among other features, the instrument allows multiple pass cyclic ion mobility separation, with concomitant increase in resolving power. Single-pass experiments enabled the detection of 30 proteins from mouse brain tissue, rising to 44 when quadrupole isolation was employed. In the absence of ion mobility separation, 21 proteins were detected in rat kidney tissue including the abundant α- and β-globin chains from hemoglobin. Single-pass cyclic ion mobility mass spectrometry enabled the detection of 60 additional proteins. Multipass experiments of a narrow m/z range (m/z 870–920) resulted in the detection of 24 proteins (one pass), 37 proteins (two passes) and 54 proteins (three passes), thus demonstrating the benefits of improved mobility resolving power.

Rhapso: Automatic Stitching of Mass Segments from Fourier Transform Ion Cyclotron Resonance Mass Spectra.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the resolution and mass accuracy needed to analyze complex mixtures such as crude oil. When mixtures contain many different components, a competitive effect within the ICR cell takes place that hampers the detection of a potentially large fraction of the components. Recently, a new data collection technique, which consists of acquiring several spectra of small mass ranges and assembling a complete spectrum afterward, enabled the observation of a record number of peaks with greater accuracy compared to broadband methods. There is a need for statistical methods to combine and preprocess segmented acquisition data. A particular challenge of quadrupole isolation is that near the window edges there is a drop in intensity, hampering the stitching of consecutive windows. We developed an algorithm called Rhapso to stitch peak lists corresponding to multiple different m/z regions from crude oil samples. Rhapso corrects potential edge effects to enable the use of smaller windows and reduce the required overlap between windows, corrects mass shifts between windows, and generates a single peak list for the full spectrum. Relative to a stitching performed manually, Rhapso increased the data processing speed and avoided potential human errors, simplifying the subsequent chemical analysis of the sample. Relative to a broadband spectrum, the stitched output showed an over 2-fold increase in assigned peaks and reduced mass error by a factor of 2. Rhapso is expected to enable routine use of this spectral stitching method for ultracomplex samples, giving a more detailed characterization of existing samples and enabling the characterization of samples that were previously too complex to analyze.

Native Electron Capture Dissociation Maps to Iron-Binding Channels in Horse Spleen Ferritin.

Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.

Additional precursor purification in isobaric mass tagging experiments by traveling wave ion mobility separation (TWIMS).

Despite the increasing popularity of data-independent acquisition workflows, data-dependent acquisition (DDA) is still the prevalent method of LC-MS-based proteomics. DDA is the basis of isobaric mass tagging technique, a powerful MS2 quantification strategy that allows coanalysis of up to 10 proteomics samples. A well-documented limitation of DDA, however, is precursor coselection, whereby a target peptide is coisolated with other ions for fragmentation. Here, we investigated if additional peptide purification by traveling wave ion mobility separation (TWIMS) can reduce precursor contamination using a mixture of Saccharomyces cerevisiae and HeLa proteomes. In accordance with previous reports on FAIMS-Orbitrap instruments, we find that TWIMS provides a remarkable improvement (on average 2.85 times) in the signal-to-noise ratio for sequence ions. We also report that TWIMS reduces reporter ions contamination by around one-third (to 14-15% contamination) and even further (to 6-9%) when combined with a narrowed quadrupole isolation window. We discuss challenges associated with applying TWIMS purification to isobaric mass tagging experiments, including correlation between ion m/z and drift time, which means that coselected peptides are expected to have similar mobility. We also demonstrate that labeling results in peptides having more uniform m/z and drift time distributions than observed for unlabeled peptides. Data are available via ProteomeXchange with identifier PXD001047.

Technical considerations for large-scale parallel reaction monitoring analysis

Targeted methods have gained acceptance among proteomics community to perform quantitative experiments. However, the current reference to conduct such experiments relies on selected reaction monitoring (SRM) analyses performed on triple quadrupole mass spectrometers, although it suffers from some limitations. First, the low resolution quadrupole mass analyzers do not present enough selectivity to discriminate the analytes from interferences commonly encountered in biological samples. Second, the number of peptides monitored in one single experiment often remains limited. The introduction of high resolution/accurate mass instruments with fast sequencing capabilities has enabled the development of novel quantitative methods. More specifically, the new quadrupole-orbitrap mass spectrometer operated in parallel reaction monitoring (PRM) mode showed detection and quantification performances similar or better than those obtained in SRM, due to the increased selectivity of the high-resolution orbitrap mass analyzer. The versatility of the instrument, with its ability to multiplex the selection of precursor ions and to operate with varying quadrupole isolation windows, has enabled the design of large-scale experiments, which require the optimization of several acquisition parameters to maintain high performance. It includes the adjustments of the fill time of the trapping device and the tight scheduling of elution times of the peptides, ideally adjusted on-the-fly. The present study constitutes a valuable baseline for the proteomics community to better control the trade-off between sensitivity and number of analyzed peptides in large-scale parallel reaction monitoring (PRM) experiments performed on a quadrupole-orbitrap instrument. A standard acquisition method requires careful setting of the parameters, namely the fill time in accordance with the control of the resolving power and the degree of multiplexing on one hand, and the quadrupole selection window on the other hand. This study helps in establishing acquisition parameters for large-scale PRM experiments, while maintaining sufficient sensitivity. In addition, the real-time correction of the scheduled peptide monitoring windows, to compensate for possible elution time drift, was explored. This approach supports the use of narrowed monitoring windows in PRM analyses, which greatly scale up the number of peptides targeted in a single LC-MS experiment. The broad application of the presented approaches by the community is likely to allow the establishment of an unprecedented scale for targeted proteomics, thus matching the pressing demand of systems biology or biomarker evaluation studies. This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

The One Hour Yeast Proteome

We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (x̄) peptide spectral matches and 34,255 (x̄) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.

Characterization of heavy petroleum fraction by positive-ion electrospray ionization FT-ICR mass spectrometry and collision induced dissociation: Bond dissociation behavior and aromatic ring architecture of basic nitrogen compounds

This paper examined the bond dissociation behavior and aromatic ring architecture of basic nitrogen compounds in Sudan heavy petroleum fraction. Both broadband and quadrupole isolation modes positive-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) coupled with collision induced dissociation (CID) techniques were used to characterize a low sulfur crude oil derived vacuum residuum (VR). The appropriate CID operating condition was selected by comparing the molecular weight distributions of the basic nitrogen compounds under various CID operating conditions. Both odd- and even-electron fragment ions were observed from the mass spectrum, indicating that the heterolytic and homolytic bond cleavages occurred simultaneously during the CID process. The odd-electron fragment ions were predominant in each class species, indicating preferential heterolytic bond cleavages. At the optimal CID condition, the alkyl groups decomposed deeply and just left the aromatic cores of the nitrogen compounds. No significant variation in double bond equivalent (DBE) value was observed between the fragment and parent ions, revealing that the domination of single core structure.

Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

Selectivity of LC-MS/MS analysis: Implication for proteomics experiments

The recent development of hybrid mass spectrometers with high resolution and accurate mass capabilities has opened new avenues in quantitative proteomics. A systematic study was performed to assess the quantification performances of a novel quadrupole-Orbitrap instrument operated in MS/MS mode (parallel reaction monitoring). It included the analyses of 35 isotopically labeled peptides spiked in urine samples to establish their dilution curves. The results were evaluated by replicating the analyses on a triple quadrupole instrument operated in selected reaction monitoring (SRM; often referred as multiple reaction monitoring, MRM) mode to assess and compare the gain in selectivity resulting from high resolution fragment ion analysis. The high resolving power dramatically increased the selectivity of measurements by separating ions of interest from interferences, which occurred in several cases, and thus improved the quantification performance. In addition, an experiment to assess the "co-habitation" of fragment ions in specific regions of the LC-MS/MS spectral space of a complex proteome digest was carried out. The study included the evaluation of the fragmentation patterns acquired under various experimental conditions (i.e., quadrupole isolation windows and Orbitrap resolving powers) for more than 200 peptides, which provided an experimental baseline to guide the development of methods for parallel reaction monitoring acquisition. This article is part of a Special Issue entitled: From protein structures to clinical applications.