Isotopic Envelopes Research Articles

Complete resolution across the neodymium/samarium isotopic envelope with a liquid sampling-atmospheric pressure glow discharge - Orbitrap mass spectrometer.

Nd and Sm isotope ratios play an important role in geological dating and as nuclear forensic signatures; however, the overlap of the respective 144, 148, 150 Nd/Sm isobars requires prior separations to be performed before analysis on typical MS platforms. The work presented here overcomes these isobaric interferences using ultrahigh-mass resolution to alleviate interference without prior chemical separations. A liquid sampling-atmospheric pressure glow discharge ion source was coupled to a standard, QExactive Focus Orbitrap mass spectrometer, providing a mass resolution of ~80 k. A Spectroswiss FTMS booster X2 data acquisition package was used to collect extended transients, providing much higher mass resolution; ~230 k and ~600 k are employed here for Nd and Sm isotopes. While the standard Orbitrap resolution is far greater than typical "atomic" MS platforms, it was insufficient to alleviate all isobars. The use of a resolution of ~230 k resulted in baseline separation across the entire isotopic envelope for both Nd and Sm. Isotope ratios obtained from Nd:Sm mixtures using high-resolution were equivalent to those found for individual-element solutions, while isotope ratios obtained at a resolution of ~80 k (standard for the OEM data system) showed large deviations. Use of ultrahigh-resolution is an attractive alternative to extensive chemical separations to alleviate severe isobaric interferences. Sufficient mass resolution greatly reduces/eliminates the need for sample manipulations (separations) before analysis while reducing costs and total analysis times.

FREE: Enhanced Feature Representation for Isotopic Envelope Evaluation in Top-Down Mass Spectra Deconvolution.

The aim of deconvolution of top-down mass spectra is to recognize monoisotopic peaks from the experimental envelopes in raw mass spectra. So accurate assessment of similarity between theoretical and experimental envelopes is a critical step in mass spectra data deconvolution. Existing evaluation methods primarily rely on intensity differences and m/z similarity, potentially lacking a comprehensive assessment. To overcome this constraint and facilitate a comprehensive and refined assessment of the similarity between theoretical and experimental envelopes, there exists an imperative to systematically explore and identify increasingly efficacious features for assessing this correspondence. We present enhanced feature representation for isotopic envelope evaluation (FREE) that derives diverse feature representations, encapsulating fundamental physical attributes of envelopes, including peak intensity and envelope shape. We trained FREE and evaluated its performance on both the ovarian tumor (OT) (human OT cells) data set and zebrafish (ZF) (brain in mature female ZF) data set. Specifically, comparing the state-of-art method, FREE demonstrates higher performance in multiple evaluation metrics across both the OT and ZF data sets, with a particular emphasis on precision, and it demonstrates accurate predictions of a greater number of positive envelopes among the top-ranked envelopes based on their scores. Moreover, within a cross-species data set of ZF, FREE identified a higher number of proteoform-spectrum matches (PrSMs), increasing the count from 50,795 to 52,927 compared to EnvCNN, the amalgamation of FREE with TopFD also exhibits a commendable capacity to discern 117,883 fragment ions, thus surpassing the 97,554 fragment ions identified through the application of EnvCNN in conjunction with TopFD. To further validate the performance of FREE, we have tested 10 a cross-species top-down proteomes containing 36 subdata set from ProteomeXchange. The results reveal that, after deconvolution with TopFD + FREE, TopPIC identifies more PrSMs across these 10 data sets in both the first and second rounds of experiments. These findings underscore the robustness and generalization capabilities of the FREE approach in diverse proteomes.

Improved determination of protein turnover rate with heavy water labeling by mass isotopomer ratio selection.

We describe a reverse lookup method to calculate the molar fraction of new synthesis from numerically approximated peptide isotopomer profiles in heavy water labeling mass spectrometry experiments. Using an experimental calibration curve comprising mixtures of fully unlabeled and fully labeled proteomes at various proportions, we show that this method provides a straightforward way to calculate the proportion of new proteins in a protein pool from arbitrarily chosen isotopomer ratios. We next analyzed which of the isotopomer pairs within the peptide isotope envelope yielded isotopomer time courses that fit most closely to kinetic models, and found that the identity of the isotopomer pair depends partially on the number of deuterium accessible labeling sites of the peptide. We next derived a strategy to automatically select the isotopomer pairs to calculate turnover rates based on peptide sequence, and showed that this increases the coverage of existing proteome-wide turnover experiments in multiple data sets of the mouse heart, liver, kidney, and skeletal muscle by up to 25-82%.

Maximizing Glycoproteomics Results through an Integrated Parallel Accumulation Serial Fragmentation Workflow.

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics has evolved rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of parallel accumulation serial fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, the reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we developed an integrated methodology using timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width, and the use of dopant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings, showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 min, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics conditions to up to 1500 with our optimized glycoproteomics approach, highlighting the need for tailored optimizations to obtain comprehensive data.

Metabolite profiling of chemically modified oligonucleotides in rat liver homogenates using hydrophilic interaction liquid chromatography-high resolution mass spectrometry

Chemically modified oligonucleotide is a rapidly emerging field of biotherapeutics. The evaluation of the metabolic stability of therapeutic oligonucleotides is critical. The current methods are limited to the ion-pairing reversed phase chromatographic techniques. Due to complex chemical modifications of therapeutic oligonucleotides and diverse structural changes in their metabolites, comprehensive metabolite profiling is still challenging. Therefore, an alternative analytical method for the metabolism study of oligonucleotides is urgently needed. In this study, a novel hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) method was developed and applied to the metabolism study of two chemically modified oligonucleotides, i.e., N11-GalNAc (phosphorothioate, 2′-F, 2′-OMe, and 3′-GalNAc) and N9 (phosphorothioate, 2′-F), in rat liver homogenates. Highly broad retention of metabolites on the HILIC column was achieved, with truncated metabolite having a single nucleotide being detected. A stepped normalized collisional energy (NCE) in HRMS led to characteristic c/y, d/y, and b/y fragment ion series, and diagnostic fragment ions for linker and GalNAc, which covered the whole sequence of oligonucleotides. A total number of twenty-seven and twenty-nine metabolites were identified in rat liver homogenates for N11-GalNAc and N9, respectively, by using the isotopic envelope of the precursor ion and stepped NCE-based sequence mapping data. The time-dependent production of metabolites was observed. The UCU sequence with the uniform 2′-F modification on ribose was identified as the major metabolic site for N11-GalNAc, and the phosphodiester bonds between nucleosides involving unmodified uracil were identified as the metabolic soft sites for N9. The developed HILIC-HRMS method provided a high coverage of various oligonucleotide metabolites. Characteristic fragment ions enable sequence mapping and therefore unambiguous metabolite identifications. This approach can be broadly applicable to the metabolism study of chemically modified oligonucleotides and the identification of their metabolic soft sites.

Ultrafast Reaction of the Drug Hydralazine with Apurinic/Apyrimidinic Sites in DNA Gives Rise to a Stable Triazolo[3,4-a]phthalazine Adduct.

The clinically used antihypertensive agent hydralazine rapidly generates hydrazone-derived adducts by reaction with apurinic/apyrimidinic (also known as abasic or AP) sites in many different sequences of duplex DNA. The reaction rates are comparable to those of some AP-trapping reagents previously described as "ultrafast." Initially, reversible formation of a hydrazone adduct is followed by an oxidative cyclization reaction that generates a chemically stable triazolo[3,4-a]phthalazine adduct. The net result is that the reaction of hydralazine with AP sites in duplex DNA yields a rapid and irreversible adduct formation. Although the hydrazone and triazolo[3,4-a]phthalazine adducts differ by only two mass units, it was possible to use MALDI-TOF-MS and ESI-QTOF-nanospray-MS to quantitatively characterize mixtures of these adducts by deconvolution of overlapping isotope envelopes. Reactions of hydralazine with the endogenous ketone pyruvate do not prevent the formation of the hydralazine-AP adducts, providing further evidence that these adducts have the potential to form in cellular DNA. AP sites are ubiquitous in cellular DNA, and rapid, irreversible adduct formation by hydralazine could be relevant to the pathogenesis of systemic drug-induced lupus erythematosus experienced by some patients. Finally, hydralazine might be developed as a probe for the detection of AP sites, the study of cellular BER, and marking the location of AP sites in DNA-sequencing analyses.

Spectral averaging with outlier rejection algorithms to increase identifications in top-down proteomics.

The identification of proteoforms by top-down proteomics requires both high quality fragmentation spectra and the neutral mass of the proteoform from which the fragments derive. Intact proteoform spectra can be highly complex and may include multiple overlapping proteoforms, as well as many isotopic peaks and charge states. The resulting lower signal-to-noise ratios for intact proteins complicates downstream analyses such as deconvolution. Averaging multiple scans is a common way to improve signal-to-noise, but mass spectrometry data contains artifacts unique to it that can degrade the quality of an averaged spectra. To overcome these limitations and increase signal-to-noise, we have implemented outlier rejection algorithms to remove outlier measurements efficiently and robustly in a set of MS1 scans prior to averaging. We have implemented averaging with rejection algorithms in the open-source, freely available, proteomics search engine MetaMorpheus. Herein, we report the application of the averaging with rejection algorithms to direct injection and online liquid chromatography mass spectrometry data. Averaging with rejection algorithms demonstrated a 45% increase in the number of proteoforms detected in Jurkat T cell lysate. We show that the increase is due to improved spectral quality, particularly in regions surrounding isotopic envelopes.

Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

Identification of sulfhydryl-containing proteins and further evaluation of the selenium-tagged redox homeostasis-regulating proteins

Chemoproteomic profiling of sulfhydryl-containing proteins has consistently been an attractive research hotspot. However, there remains a dearth of probes that are specifically designed for sulfhydryl-containing proteins, possessing sufficient reactivity, specificity, distinctive isotopic signature, as well as efficient labeling and evaluation capabilities for proteins implicated in the regulation of redox homeostasis. Here, the specific selenium-containing probes (Se-probes) in this work displayed high specificity and reactivity toward cysteine thiols on small molecules, peptides and purified proteins and showed very good competitive effect of proteins labeling in gel-ABPP. We identified more than 6000 candidate proteins. In TOP-ABPP, we investigated the peptide labeled by Se-probes, which revealed a distinct isotopic envelope pattern of selenium in both the primary and secondary mass spectra. This unique pattern can provide compelling evidence for identifying redox regulatory proteins and other target peptides. Furthermore, our examiation of post-translational modification (PTMs) of the cysteine site residues showed that oxidation PTMs was predominantly observed. We anticipate that Se-probes will enable broader and deeper proteome-wide profiling of sulfhydryl-containing proteins, provide an ideal tool for focusing on proteins that regulate redox homeostasis and advance the development of innovative selenium-based pharmaceuticals.

High-Definition Ion Mobility/Mass Spectrometry with Structural Isotopic Shifts for Nominally Isobaric Isotopologues

We had reported the isotopic envelopes in differential IMS (FAIMS) separations depending on the ion structure. However, this new approach to distinguish isomers was constrained by the unit-mass resolution commingling all nominally isobaric isotopologues. Here, we directly couple high-definition FAIMS to ultrahigh-resolution (Orbitrap) MS and employ the resulting platform to explore the FAIMS spectra for isotopic fine structure. The peak shifts therein for isotopologues of halogenated anilines with 15N and 13C (split by 6 mDa) in N2/CO2 buffers dramatically differ, more than for the 13C, 37Cl, or 81Br species apart by 1 or 2 Da. The shifts in FAIMS space upon different elemental isotopic substitutions are orthogonal mutually and to the underlying separations, forming fingerprint multidimensional matrices and 3-D trajectories across gas compositions that redundantly delineate all isomers considered. The interlocking instrumental and methodological upgrades in this work take the structural isotopic shift approach to the next level.

Parchment Glutamine Index (PQI): A novel method to estimate glutamine deamidation levels in parchment collagen obtained from low-quality MALDI-TOF data

Parchment was used as a writing material in the Middle Ages and was made using animal skins by liming them with Ca(OH)2. During liming, collagen peptides containing Glutamine (Q) undergo deamidation resulting in a mass shift of 0.984 Da. Assessing the extent of deamidation can inform us about parchment production patterns and quality. In this study, we propose a simple three-step workflow, developed as an R package called MALDIpqi(), to estimate deamidation in parchment derived collagen using low-resolution MALDI-TOF spectra. After pre-processing raw spectra, we used weighted least-squares linear regression to estimate Q deamidation levels from the convoluted isotopic envelope for seven collagen-peptide markers. Finally, we employed a linear mixed effect model to predict the overall deamidation level of a parchment sample termed Parchment Glutamine Index (PQI). To test the robustness of the workflow, we applied MALDIpqi() to previously published ZooMS data generated from almost an entire library of the Cistercian monastery at Orval Abbey, Belgium. In addition to reliably predicting PQI, we observed interesting patterns pertaining to parchment production. MALDIpqi() holds excellent potential for biocodicological and other archaeological studies involving collagen, such as bone, but we also foresee its application in the food and biomedical industry.

Envemind: Accurate Monoisotopic Mass Determination Based On Isotopic Envelope

Nowadays, monoisotopic mass is used as an important feature in top-down proteomics. Knowing the exact monoisotopic mass is helpful for precise and quick protein identification in large protein databases. However, only in spectra of small molecules the monoisotopic peak is visible. For bigger molecules like proteins, it is hidden in noise or undetected at all, and therefore its position has to be predicted. By improving the prediction of the peak, we contribute to a more accurate identification of molecules, which is crucial in fields such as chemistry and medicine. In this work, we present the envemind algorithm, which is a two-step procedure to predict monoisotopic masses of proteins. The prediction is based on an isotopic envelope. Therefore, envemind is dedicated to spectra where we are able to resolve the one dalton separated isotopic variants. Furthermore, only single-molecule spectra are allowed, that is, spectra that do not require prior deconvolution. The algorithm deals with the problem of off-by-one dalton errors, which are common in monoisotopic mass prediction. A novel aspect of this work is a mathematical exploration of the space of molecules, where we equate chemical formulas and their theoretical spectrum. Since the space of molecules consists of all possible chemical formulas, this approach is not limited to known substances only. This makes optimization processes faster and enables to approximate theoretical spectrum for a given experimental one. The algorithm is available as a Python package envemind on our GitHub page https://github.com/PiotrRadzinski/envemind.

Isomer and Conformer-Specific Mass Distribution-Based Isotopic Shifts in High-Resolution Cyclic Ion Mobility Separations.

Herein, we present the use of mass distribution-based isotopic shifts in high-resolution cyclic ion mobility spectrometry-mass spectrometry (cIMS-MS)-based separations to characterize various isomeric species as well as conformers. Specifically, by using the observed relative arrival time values for the isotopologues found in the isotopic envelope after long pathlength cIMS-MS separations, we were able to distinguish dibromoaniline, dichloroaniline, and quaternary ammonium salt isomers, as well as a pair of 25-hydroxyvitamin D3 conformers based on their respective mass distribution-based shifts. Our observed shifts were highly reproducible and broadly applied to the isotopologues of various atoms (i.e., Cl, Br, and C). Additionally, through a control experiment, we determined that such shifts are indeed pathlength-independent, thus demonstrating that our presented methodology could be readily extended to other high-resolution IMS-MS platforms. These results are the first characterization of conformers using mass distribution-based IMS-MS shifts, as well as the first use of a commercial cIMS-MS platform to characterize isomers via their mass distribution-based shifts. We anticipate that our methodology will have broad applicability for biological analytes and that mass distribution-based shifts could potentially act as an added dimension of analysis in existing IMS-MS workflows in omics-based research. Specifically, we envision that the development of a database of these mass distribution-based shifts could, for example, enable the identification of unknown metabolites in complex matrices.

High-Resolution Hydrogen-Deuterium Protection Factors from Sparse Mass Spectrometry Data Validated by Nuclear Magnetic Resonance Measurements.

Experimental measurement of time-dependent spontaneous exchange of amide protons with deuterium of the solvent provides information on the structure and dynamical structural variation in proteins. Two experimental techniques are used to probe the exchange: NMR, which relies on different magnetic properties of hydrogen and deuterium, and MS, which exploits the change in mass due to deuteration. NMR provides residue-specific information, that is, the rate of exchange or, analogously, the protection factor (i.e., the unitless ratio between the rate of exchange for a completely unstructured state and the observed rate). MS provides information that is specific to peptides obtained by proteolytic digestion. The spatial resolution of HDX-MS measurements depends on the proteolytic pattern of the protein, the fragmentation method used, and the overlap between peptides. Different computational approaches have been proposed to extract residue-specific information from peptide-level HDX-MS measurements. Here, we demonstrate the advantages of a method recently proposed that exploits self-consistency and classifies the possible sets of protection factors into a finite number of alternative solutions compatible with experimental data. The degeneracy of the solutions can be reduced (or completely removed) by exploiting the additional information encoded in the shape of the isotopic envelopes. We show how sparse and noisy MS data can provide high-resolution protection factors that correlate with NMR measurements probing the same protein under the same conditions.

Monitoring and Identifying Insulin-Like Growth Factor 1 Variants by Liquid Chromatography-High-Resolution Mass Spectrometry in a Clinical Laboratory.

Protein and peptide hormones often exist as sequence variants with different molecular mass. Monitoring these variants of different molecular mass by mass spectrometry using mass-to-charge (m/z) ratio that is indicative of the wild type may lead to inaccurate quantitative results. However, liquid chromatography-high-resolution mass spectrometry (LC-HRMS)-based techniques can capture these differences and provide an opportunity to resolve, or partially resolve, variant complexity. In this chapter, we describe a general approach for monitoring a set of peptide variants with similar m/z ratios and isotopic envelopes, but different in amino acid sequences. As an example, we use insulin-like growth factor-1 (IGF-1) to demonstrate a DNA database-guided approach to monitor protein variants by LC-HRMS in a clinical laboratory. The workflow is automated and therefore avoids manual calculations that are prone to human error. The method can also monitor multiple IGF-1 variants and discover new ones. It can also provide a profile of a patient's IGF-1 status and be used to explore genotype-phenotype relationships in IGF-1 variants.

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically or into the cross-linker molecules. Isotope-labeled cross-linkers have primarily utilized deuterium and rely on MS1-based quantitation of precursor ion extracted ion chromatograms. Recently the development and application of isobaric quantitative protein interaction reporter (iqPIR) cross-linkers were reported, which utilize 13C and 15N isotope labels. Quantitation is accomplished using relative fragment ion isotope abundances in tandem mass spectra. Here we describe the synthesis and initial evaluation of a multiplexed set of iqPIR molecules, allowing for up to six cross-linked samples to be quantified simultaneously. To analyze data for such cross-linkers, the two-channel mode of iqPIR quantitative analysis was adapted to accommodate any number of channels with defined ion isotope peak mass offsets. The summed ion peak intensities in the overlapping channel isotope envelopes are apportioned among the channels to minimize the difference with respect to the predicted ion isotope envelopes. The result is accurate and reproducible relative quantitation enabling direct comparison among six differentially labeled cross-linked samples. The approach described here is generally extensible for the iqPIR strategy, accommodating future iqPIR reagent design, and enables large-scale in vivo quantitative XL-MS investigation of the interactome.

Quantitative Hydrogen/Deuterium Exchange Mass Spectrometry.

This Account describes considerations for the data generation, data analysis, and data interpretation of a hydrogen/deuterium exchange-mass spectrometry (HDX-MS) experiment to have a quantitative argument. Although HDX-MS has gained its popularity as a biophysical tool, the argument from its data often remains qualitative. To generate HDX-MS data that are more suitable for a quantitative argument, the sequence coverage and sequence resolution should be optimized during the feasibility stage, and the time window coverage and time window resolution should be improved during the HDX stage. To extract biophysically meaningful values for a certain perturbation from medium-resolution HDX-MS data, there are two major ways: (i) estimating the area between the two deuterium buildup curves using centroid values with and without the perturbation when plotted against log time scale and (ii) dissecting into multiple single-exponential curves using the isotope envelopes. To have more accurate arguments for an HDX-MS perturbation study, (i) false negatives due to sequence coverage, (ii) false negatives due to time window coverage, (iii) false positives due to sequence resolution, and (iv) false positives due to allosteric effects should be carefully examined.

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics.

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson's r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments.

Isotopic Distribution Calibration for Mass Spectrometry.

Mass spectrometry (MS) is widely used in science and industry. It allows accurate, specific, sensitive, and reproducible detection and quantification of a huge range of analytes. Across MS applications, quantification by MS has grown most dramatically, with >50 million experiments/year in the USA alone. However, quantification performance varies between instruments, compounds, different samples, and within- and across runs, necessitating normalization with analyte-similar internal standards (IS) and use of IS-corrected multipoint external calibration curves for each analyte, a complicated and resource-intensive approach, which is particularly ill-suited for multi-analyte measurements. We have developed an internal calibration method that utilizes the natural isotope distribution of an IS for a given analyte to provide internal multipoint calibration. Multiple isotope distribution calibrators for different targets in the same sample facilitate multiplex quantification, while the emerging random-access automated MS platforms should also greatly benefit from this approach. Finally, isotope distribution calibration allows mathematical correction for suboptimal experimental conditions. This might also enable quantification of hitherto difficult, or impossible to quantify, targets, if the distribution is adjusted in silico to mimic the analyte. The approach works well for high resolution, accurate mass MS for analytes with at least a modest-sized isotopic envelope. As shown herein, the approach can also be applied to lower molecular weight analytes, but the reduction in calibration points does reduce quantification performance.

Two-Dimensional Partial Covariance Mass Spectrometry for the Top-Down Analysis of Intact Proteins.

Two-dimensional partial covariance mass spectrometry (2D-PC-MS) exploits the inherent fluctuations of fragment ion abundances across a series of tandem mass spectra, to identify correlated pairs of fragment ions produced along the same fragmentation pathway of the same parent (e.g., peptide) ion. Here, we apply 2D-PC-MS to the analysis of intact protein ions in a standard linear ion trap mass analyzer, using the fact that the fragment-fragment correlation signals are much more specific to the biomolecular sequence than one-dimensional (1D) tandem mass spectrometry (MS/MS) signals at the same mass accuracy and resolution. We show that from the distribution of signals on a 2D-PC-MS map it is possible to extract the charge state of both parent and fragment ions without resolving the isotopic envelope. Furthermore, the 2D map of fragment-fragment correlations naturally separates the products of the primary decomposition pathways of the molecular ions from those of the secondary ones. We access this spectral information using an adapted version of the Hough transform. We demonstrate the successful identification of highly charged, intact protein molecules bypassing the need for high mass resolution. Using this technique, we also perform the in silico deconvolution of the overlapping fragment ion signals from two co-isolated and co-fragmented intact proteins, demonstrating a viable new method for the concurrent mass spectrometric identification of a mixture of intact protein ions from the same fragment ion spectrum.