Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.     

iTRAQ reagent-based quantitative proteomic analysis on a linear ion trap mass spectrometer

For proteomic analysis using tandem mass spectrometry, linear ion trap instruments provide unsurpassed sensitivity but unreliably detect low mass peptide fragments, precluding their use with iTRAQ reagent-labeled samples. Although the popular LTQ linear ion trap supports analyzing iTRAQ reagent-labeled peptides via pulsed Q dissociation, PQD, its effectiveness remains questionable. Using a standard mixture, we found careful tuning of relative collision energy necessary for fragmenting iTRAQ reagent-labeled peptides, and increasing microscan acquisition and repeat count improves quantification but identifies somewhat fewer peptides. We developed software to calculate abundance ratios via summing reporter ion intensities across spectra matching to each protein, thereby providing maximized accuracy. Testing found that results closely corresponded between analysis using optimized LTQ-PQD settings plus our software and using a Qstar instrument. Thus, we demonstrate the effectiveness of LTQ-PQD analyzing iTRAQ reagent-labeled peptides, and provide guidelines for successful quantitative proteomic studies.     

In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos

Background

Hen's egg white has been the subject of intensive chemical, biochemical and food technological research for many decades, because of its importance in human nutrition, its importance as a source of easily accessible model proteins, and its potential use in biotechnological processes. Recently the arsenal of tools used to study the protein components of egg white has been complemented by mass spectrometry-based proteomic technologies. Application of these fast and sensitive methods has already enabled the identification of a large number of new egg white proteins. Recent technological advances may be expected to further expand the egg white protein inventory.

Results

Using a dual pressure linear ion trap Orbitrap instrument, the LTQ Orbitrap Velos, in conjunction with data analysis in the MaxQuant software package, we identified 158 proteins in chicken egg white with two or more sequence unique peptides. This group of proteins identified with very high confidence included 79 proteins identified in egg white for the first time. In addition, 44 proteins were identified tentatively.

Conclusions

Our results, apart from identifying many new egg white components, indicate that current mass spectrometry technology is sufficiently advanced to permit direct identification of minor components of proteomes dominated by a few major proteins without resorting to indirect techniques, such as chromatographic depletion or peptide library binding, which change the composition of the proteome.     

Characterization of a high field Orbitrap mass spectrometer for proteome analysis

The field of proteomics continues to be driven by improvements in analytical technology, notably in peptide separation, quantitative MS, and informatics. In this study, we have characterized a hybrid linear ion trap high field Orbitrap mass spectrometer (Orbitrap Elite) for proteomic applications. The very high resolution available on this instrument allows 95% of all peptide masses to be measured with sub‐ppm accuracy that in turn improves protein identification by database searching. We further confirm again that mass accuracy in tandem mass spectra is a valuable parameter for improving the success of protein identification. The new CID rapid scan type of the Orbitrap Elite achieves similar performance as higher energy collision induced dissociation fragmentation and both allow the identification of hundreds of proteins from as little as 0.1 ng of protein digest on column. The new instrument outperforms its predecessor the Orbitrap Velos by a considerable margin on each metric assessed that makes it a valuable and versatile tool for MS‐based proteomics.     

Evaluation of different peptide fragmentation types and mass analyzers in data‐dependent methods using an Orbitrap Fusion Lumos Tribrid mass spectrometer

One of the major additions in MS technology has been the irruption of the Orbitrap mass analyzer, which has boosted the proteomics analyses of biological complex samples since its introduction. Here, we took advantage of the capabilities of the new Orbitrap Fusion Lumos Tribrid mass spectrometer to assess the performance of different data‐dependent acquisition methods for the identification and quantitation of peptides and phosphopeptides in single‐shot analysis of human whole cell lysates. Our study explored the capabilities of tri‐hibrid mass spectrometers for (phospho‐) peptide identification and quantitation using different gradient lengths, sample amounts, and combinations of different peptide fragmentation types and mass analyzers. Moreover, the acquisition of the same complex sample with different acquisition methods resulted in the generation of a dataset to be used as a reference for further analyses, and a starting point for future optimizations in particular applications.     

Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer

Mass spectrometry-based proteomics has greatly benefitted from enormous advances in high resolution instrumentation in recent years. In particular, the combination of a linear ion trap with the Orbitrap analyzer has proven to be a popular instrument configuration. Complementing this hybrid trap-trap instrument, as well as the standalone Orbitrap analyzer termed Exactive, we here present coupling of a quadrupole mass filter to an Orbitrap analyzer. This "Q Exactive" instrument features high ion currents because of an S-lens, and fast high-energy collision-induced dissociation peptide fragmentation because of parallel filling and detection modes. The image current from the detector is processed by an "enhanced Fourier Transformation" algorithm, doubling mass spectrometric resolution. Together with almost instantaneous isolation and fragmentation, the instrument achieves overall cycle times of 1 s for a top 10 higher energy collisional dissociation method. More than 2500 proteins can be identified in standard 90-min gradients of tryptic digests of mammalian cell lysate- a significant improvement over previous Orbitrap mass spectrometers. Furthermore, the quadrupole Orbitrap analyzer combination enables multiplexed operation at the MS and tandem MS levels. This is demonstrated in a multiplexed single ion monitoring mode, in which the quadrupole rapidly switches among different narrow mass ranges that are analyzed in a single composite MS spectrum. Similarly, the quadrupole allows fragmentation of different precursor masses in rapid succession, followed by joint analysis of the higher energy collisional dissociation fragment ions in the Orbitrap analyzer. High performance in a robust benchtop format together with the ability to perform complex multiplexed scan modes make the Q Exactive an exciting new instrument for the proteomics and general analytical communities.     

Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation

Coupling of multiplex isobaric tags for relative and absolute quantitation (iTRAQ) to a sensitive linear ion trap (LTQ) mass spectrometer (MS) is a challenging, but highly promising approach for quantitative high-throughput proteomic profiling. Integration of the advantages of pulsed-Q dissociation (PQD) and collision-activated dissociation (CAD) fragmentation methods into a PQD-CAD hybrid mode, together with PQD optimization and data manipulation with a bioinformatics algorithm, resulted in a robust, sensitive and accurate iTRAQ quantitative proteomic workflow. The workflow was superior to the default PQD setting when profiling the proteome of a gastric cancer cell line, SNU5. Taken together, we established an optimized PQD-CAD hybrid workflow in LTQ-MS for iTRAQ quantitative proteomic profiling that may have wide applications in biological and biomedical research.     

Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes

Although only a few years old, the combination of a linear ion trap with an Orbitrap analyzer has become one of the standard mass spectrometers to characterize proteins and proteomes. Here we describe a novel version of this instrument family, the Orbitrap Elite, which is improved in three main areas. The ion transfer optics has an ion path that blocks the line of sight to achieve more robust operation. The tandem MS acquisition speed of the dual cell linear ion trap now exceeds 12 Hz. Most importantly, the resolving power of the Orbitrap analyzer has been increased twofold for the same transient length by employing a compact, high-field Orbitrap analyzer that almost doubles the observed frequencies. An enhanced Fourier Transform algorithm-incorporating phase information-further doubles the resolving power to 240,000 at m/z 400 for a 768 ms transient. For top-down experiments, we combine a survey scan with a selected ion monitoring scan of the charge state of the protein to be fragmented and with several HCD microscans. Despite the 120,000 resolving power for SIM and HCD scans, the total cycle time is within several seconds and therefore suitable for liquid chromatography tandem MS. For bottom-up proteomics, we combined survey scans at 240,000 resolving power with data-dependent collision-induced dissociation of the 20 most abundant precursors in a total cycle time of 2.5 s-increasing protein identifications in complex mixtures by about 30%. The speed of the Orbitrap Elite furthermore allows scan modes in which complementary dissociation mechanisms are routinely obtained of all fragmented peptides.     

Systematic comparison of label-free, metabolic labeling, and isobaric chemical labeling for quantitative proteomics on LTQ Orbitrap Velos

A variety of quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling using iTRAQ or TMT. Here, these methods were compared in terms of the depth of proteome coverage, quantification accuracy, precision, and reproducibility using a high-performance hybrid mass spectrometer, LTQ Orbitrap Velos. Our results show that (1) the spectral counting method provides the deepest proteome coverage for identification, but its quantification performance is worse than labeling-based approaches, especially the quantification reproducibility; (2) metabolic labeling and isobaric chemical labeling are capable of accurate, precise, and reproducible quantification and provide deep proteome coverage for quantification; isobaric chemical labeling surpasses metabolic labeling in terms of quantification precision and reproducibility; and (3) iTRAQ and TMT perform similarly in all aspects compared in the current study using a CID-HCD dual scan configuration. On the basis of the unique advantages of each method, we provide guidance for selection of the appropriate method for a quantitative proteomics study.     

Orbitrap mass analyzer--overview and applications in proteomics

The orbitrap mass analyzer is proving itself as a useful addition to a proteomics tool box. The key attributes of this analyzer are accurate mass and high resolution similar to those achievable with FT ICR instrumentation. The basic principles underlying these capabilities, and how they translate into benefits in real-life proteomics experiments are discussed. The focus is on reviewing examples of protein identification with bottom-up and top-down approaches, and detection of post-translational modifications.     

Detection and quantification of snow algae with an airborne imaging spectrometer.

We describe spectral reflectance measurements of snow containing the snow alga Chlamydomonas nivalis and a model to retrieve snow algal concentrations from airborne imaging spectrometer data. Because cells of C. nivalis absorb at specific wavelengths in regions indicative of carotenoids (astaxanthin esters, lutein, beta-carotene) and chlorophylls a and b, the spectral signature of snow containing C. nivalis is distinct from that of snow without algae. The spectral reflectance of snow containing C. nivalis is separable from that of snow without algae due to carotenoid absorption in the wavelength range from 0.4 to 0.58 microm and chlorophyll a and b absorption in the wavelength range from 0.6 to 0.7 microm. The integral of the scaled chlorophyll a and b absorption feature (I(0.68)) varies with algal concentration (C(a)). Using the relationship C(a) = 81019.2 I(0.68) + 845.2, we inverted Airborne Visible Infrared Imaging Spectrometer reflectance data collected in the Tioga Pass region of the Sierra Nevada in California to determine algal concentration. For the 5.5-km(2) region imaged, the mean algal concentration was 1,306 cells ml(-1), the standard deviation was 1,740 cells ml(-1), and the coefficient of variation was 1.33. The retrieved spatial distribution was consistent with observations made in the field. From the spatial estimates of algal concentration, we calculated a total imaged algal biomass of 16.55 kg for the 0.495-km(2) snow-covered area, which gave an areal biomass concentration of 0.033 g/m(2).     

Low energy peptide fragmentations in an ESI-Q-Tof type mass spectrometer

Efficient peptide sequencing relies on both high quality MS/MS data acquisition and exhaustive knowledge of gas-phase dissociation mechanisms. We report our contribution to the elaboration of more comprehensive fragmentation models required for efficient automated MS/MS spectra interpretation. Following a statistical approach, various peptides (296 sequences of variable compositions and lengths) were prepared and subjected to low-energy collision-induced dissociations (CID) in an electrospray hybrid instrument (ESI-Q-q-Tof type mass spectrometer) that has retained relatively limited attention so far. Besides, our studies were focused on low molecular weight singly charged peptides that often failed to be identified by sequencing algorithms. Only half of the studied compounds showed charge directed dissociations in accordance with the mobile proton model producing fragment ions directly related to the primary sequence. For the peptides that did not exhibit the expected fragment ion series, alternative dissociation behaviors issued from complex rearrangements were evidenced.     

18O-labeling quantitative proteomics using an ion trap mass spectrometer

We describe a method for simultaneous identification and quantitation of proteins within complex mixtures. The method consists of 18O-labeling, a simple stable isotope-coding that requires merely enzymatic digestion in 18O-water, in combination with a capillary-liquid chromatography electrospray ion-trap mass spectrometer. In a separate experiment using the same sample and a spike test, we demonstrate that the difference ration was calculated accurately using the 18O-labeling method even if the protein was part of a complex mixture. Our data also suggest that the accuracy of the quantitation can be improved by averaging the difference ratios of several peptides. In comparing our method with the isotope-coded affinity tag (ICAT) method, we show that the 18O-labeling method has the advantages of better recovery and fewer isotope effects. Therefore, the 18O-labeling method is a powerful tool for large-scale proteomics applications.     

A simple and sensitive liquid chromatography-tandem mass spectrometry assay for the quantification of ertapenem in microdialysate

A new liquid chromatography assay with isocratic elution and tandem mass spectrometry detection (LC-MS/MS) using an electrospray ionization interface in the multiple reaction monitoring mode was developed and validated for ertapenem determination in microdialysate samples. Linearity was demonstrated between 10ngmL(-1) (lower limit of quantification, LLoQ) and 160ngmL(-1). The precision (CV%) and accuracy (bias%) in microdialysates at the LLoQ were respectively 2.2% and 17.3% within-day and 10.6% and 2.7% between-days. Ertapenem was stable for 1 month at -20 degrees C and -80 degrees C but unstable at +4 degrees C. This new LC-MS/MS assay is simple, rapid and more sensitive than previously described assays.     

A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.     

Integrating titania enrichment,iTRAQ labeling,and Orbitrap CID‐HCD for global identification and quantitative analysis of phosphopeptides

Recent advances in MS instrumentation and progresses in phosphopeptide enrichment, in conjunction with more powerful data analysis tools, have facilitated unbiased characterization of thousands of site‐specific phosphorylation events. Combined with stable isotope labeling by amino acids in cell culture metabolic labeling, these techniques have made it possible to quantitatively evaluate phosphorylation changes in various physiological states in stable cell lines. However, quantitative phosphoproteomics in primary cells and tissues remains a major technical challenge due to the lack of adequate techniques for accurate quantification. Here, we describe an integrated strategy allowing for large scale quantitative profiling of phosphopeptides in complex biological mixtures. In this technique, the mixture of proteolytic peptides was subjected to phosphopeptide enrichment using a titania affinity column, and the purified phosphopeptides were subsequently labeled with iTRAQ reagents. After further fractionation by strong‐cation exchange, the peptides were analyzed by LC‐MS/MS on an Orbitrap mass spectrometer, which collects CID and high‐energy collisional dissociation (HCD) spectra sequentially for peptide identification and quantitation. We demonstrate that direct phosphopeptide enrichment of protein digests by titania affinity chromatography substantially improves the efficiency and reproducibility of phosphopeptide proteomic analysis and is compatible with downstream iTRAQ labeling. Conditions were optimized for HCD normalized collision energy to balance the overall peptide identification and quantitation using the relative abundances of iTRAQ reporter ions. Using this approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell lysates, of which 2709 were also quantified from HCD scans.     

Automated identification and quantification of protein phosphorylation sites by LC/MS on a hybrid triple quadrupole linear ion trap mass spectrometer

Complete phosphorylation mapping of protein kinases was successfully undertaken using an automated LC/MS/MS approach. This method uses the direct combination of triple quadrupole and ion trapping capabilities in a hybrid triple quadrupole linear ion trap to selectively identify and sequence phosphorylated peptides. In particular, the use of a precursor ion scan of m/z -79 in negative ion mode followed by an ion trap high resolution scan (an enhanced resolution scan) and a high sensitivity MS/MS scan (enhanced product ion scan) in positive mode is a very effective method for identifying phosphorylation sites in proteins at low femtomole levels. Coupling of this methodology with a stable isotope N-terminal labeling strategy using iTRAQtrade mark reagents enabled phosphorylation mapping and relative protein phosphorylation levels to be determined between the active and inactive forms of the protein kinase MAPKAPK-1 in the same LC/MS run.     

A comparison of the accuracy of iTRAQ quantification by nLC-ESI MSMS and nLC-MALDI MSMS methods

The accuracy of quantification obtained using the iTRAQ labelling methodology for measuring protein ratios more extreme than 1:1 was investigated. A comparison of nLC-ESI MSMS and nLC-MALDI MSMS analysis routes was performed. A fixed concentration of a standard six protein mix was spiked with two proteins at a range of concentrations. The two data analysis programmes, Mascot and ProteinPilot Paragon, were also compared. Whilst the lower ratios could be measured accurately, greater discrepancies were seen for the higher ratios, particularly by nLC-ESI MSMS. Filtering out the weaker reporter ion signals improved the accuracy of the ratios: this is likely due to several factors which are explored in more detail. Overall, analysis by nLC-MALDI MSMS followed by Mascot interpretation gave the most accurate results.     

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of mometasone furoate in human plasma

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC-(APCI(-))-MS-MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were < or = 15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.