Precursor Fragmentation Research Articles

Expanding Proteoform Identifications in Top-Down Proteomic Analyses by Constructing Proteoform Families.

In top-down proteomics, intact proteins are analyzed by tandem mass spectrometry and proteoforms, which are defined forms of a protein with specific sequences of amino acids and localized post-translational modifications, are identified using precursor mass and fragmentation data. Many proteoforms that are detected in the precursor scan (MS1) are not selected for fragmentation by the instrument and therefore remain unidentified in typical top-down proteomic workflows. Our laboratory has developed the open source software program Proteoform Suite to analyze MS1-only intact proteoform data. Here, we have adapted it to provide identifications of proteoform masses in precursor MS1 spectra of top-down data, supplementing the top-down identifications obtained using the MS2 fragmentation data. Proteoform Suite performs mass calibration using high-scoring top-down identifications and identifies additional proteoforms using calibrated, accurate intact masses. Proteoform families, the set of proteoforms from a given gene, are constructed and visualized from proteoforms identified by both top-down and intact-mass analyses. Using this strategy, we constructed proteoform families and identified 1861 proteoforms in yeast lysate, yielding an approximately 40% increase over the original 1291 proteoform identifications observed using traditional top-down analysis alone.

Deposition of fluorocarbon groups on wood surfaces using the jet of an atmospheric-pressure dielectric barrier discharge

This work reports the functionalization of sugar maple (Acer saccharum) wood surfaces following their exposure to the jet of an atmospheric-pressure dielectric barrier discharge operated in either Ar or N2 with C3F8 (octafluoropropane) as the growth precursor for plasma-enhanced chemical vapour deposition. Analysis of the current–voltage characteristics (I–V) revealed a filamentary behaviour in all conditions. While more intense current peaks were observed after addition of C3F8 in Ar, a decrease in the discharge current was seen in N2/C3F8 plasmas. Optical emission spectroscopy showed the apparition of a CF2 + continuum and CF2 rovibrational bands between 225 and 350 nm with increasing applied voltage and precursor concentration in the Ar plasmas; a feature ascribed to the presence of plasma-generated C3F8 fragments. In such conditions, water contact angle (WCA) measurements indicated that freshly sanded wood samples became highly hydrophobic, with WCAs in the 140° range and excellent dimensional stability for a period of up to 125 days. X-ray photoelectron spectroscopy revealed that such behaviours result from the deposition of highly hydrophobic CF x groups on the surface. On the other hand, no increase in surface hydrophobicity was observed following exposure to the jet of N2/C3F8 plasmas, probably due to their much lower precursor fragmentation levels.

Effect of peptide assay library size and composition in targeted data-independent acquisition-MS analyses.

The quantification of peptides using targeted analysis of data-independent acquisition MS (DIA-MS) is dependent on the size and characteristics of the assay library. We addressed several important questions on how library composition influences: (1) the number of peptides extracted from DIA-MS datasets, (2) the quality of these peptides and proteins, and (3) the biological conclusions inferred. To answer these questions we constructed five libraries from mouse vascular smooth muscle cell (VSMC) lysate, each unique in depth, input sample complexity, data acquisition mode (DDA-MS or DIA-MS), and precursor fragmentation mode (TOF-CID or Orbitrap HCD) and extracted them against the same eight DIA-MS files of VSMCs treated with vehicle or transforming growth factor β-1 (TGF-β1). We found that along with differences in peptide and protein composition, the fragments representing a given peptide differed between the libraries. Collectively these differences impacted both peak group score profile and protein abundance estimates. Surprisingly, there was little overlap in the TGF-β1 response proteome between libraries. We conclude that additional work is needed to optimize peptide assay library building for DIA-MS applications, particularly in terms of selecting optimal peptides and their respective fragments for protein quantification.

Deposition kinetics and characterization of stable ionomers from hexamethyldisiloxane and methacrylic acid by plasma enhanced chemical vapor deposition

A novel ionomer of hexamethyldisiloxane and methacrylic acid was synthesized by plasma enhanced chemical vapor deposition (PECVD). The PECVD process, being solventless, allows mixing of monomers with very different solubilities, and for polymers formed at high deposition rates and with high structural stability (due to the high number of cross-links and covalent bonding to the substrate) to be obtained. A kinetic study over a large set of parameters was run with the aim of determining the optimal conditions for high stability and proton conductivity of the polymer layer. Copolymers with good stability over 6 months' time in air and water were obtained, as demonstrated by ellipsometry, X-Ray reflectivity, and FT-IR spectroscopy. Stable coatings showed also proton conductivity as high as 1.1 ± 0.1 mS cm−1. Chemical analysis showed that due to the high molecular weight of the chosen precursors, it was possible to keep the plasma energy-input-per-mass low. This allowed limited precursor fragmentation and the functional groups of both monomers to be retained during the plasma polymerization.

Label-free quantification in ion mobility-enhanced data-independent acquisition proteomics.

Unbiased data-independent acquisition (DIA) strategies have gained increased popularity in the field of quantitative proteomics. The integration of ion mobility separation (IMS) into DIA workflows provides an additional dimension of separation to liquid chromatography-mass spectrometry (LC-MS), and it increases the achievable analytical depth of DIA approaches. Here we provide a detailed protocol for a label-free quantitative proteomics workflow based on ion mobility-enhanced DIA, which synchronizes precursor ion drift times with collision energies to improve precursor fragmentation efficiency. The protocol comprises a detailed description of all major steps including instrument setup, filter-aided sample preparation, LC-IMS-MS analysis and data processing. Our protocol can handle proteome samples of any complexity, and it enables a highly reproducible and accurate precursor intensity-based label-free quantification of up to 5,600 proteins across multiple runs in complete cellular lysates. Depending on the number of samples to be analyzed, the protocol takes a minimum of 3 d to complete from proteolytic digestion to data evaluation.

Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography.

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the author's laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed.

Applying Arginylation for Bottom-Up Proteomics.

Arginylation is an enzymatic reaction in which arginyl-tRNA protein transferase 1 (ATE1, EC 2.3.2.8) conjugates a single arginyl moiety from aminoacylated tRNA(Arg) onto a target polypeptide. We established arginylation for in vitro labeling of peptides with N-terminal acidic amino acids. Consistent with prior knowledge, arginylated peptides flanked by basic amino acids result in rich redundant MS/MS fragment spectra using various precursor fragmentation modes. Arginylation carried out by ATE1 is a fast method for labeling peptides. Sequence-specific proteolytic digest of proteins is best carried out using a double digest of proteins by Lys-C and Asp-N to generate peptides with a basic amino acid on the C-terminus and an acidic amino acid on the N-terminus. Under these conditions, arginylation is specific for N-terminal acidic amino acids and results in a near 2× sequence coverage in the MS/MS spectrum are achieved.

Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics

We present a data-independent acquisition mass spectrometry method, ultradefinition (UD) MS(E). This approach utilizes ion mobility drift time-specific collision-energy profiles to enhance precursor fragmentation efficiency over current MS(E) and high-definition (HD) MS(E) data-independent acquisition techniques. UDMS(E) provided high reproducibility and substantially improved proteome coverage of the HeLa cell proteome compared to previous implementations of MS(E), and it also outperformed a state-of-the-art data-dependent acquisition workflow. Additionally, we report a software tool, ISOQuant, for processing label-free quantitative UDMS(E) data.

Surface Functionalization of COC Microfluidic Materials by Plasma and Click Chemistry Processes

AbstractA robust method for COC surfaces functionalization was developed for the first time by plasma polymerization. 1‐bromopropane in the vapor phase allowed the formation of a brominated deposit on COC surfaces, which stability, homogeneity and chemical nature were evaluated for different experimental conditions. The analysis of vapor phase was achieved by mass spectrometry to control brominated precursor fragmentation. Following nucleophilic exchange in the presence of NaN3 to convert the brominated surface to azide‐bearing surface, these modified COC were further functionalized with a fluorescent alkyne, via the copper‐catalyzed azide–alkyne cycloaddition reaction, i.e. “click” reaction. Surface modifications were characterized by water contact angle measurement, ellipsometry, electrochemical microscopy, XPS, IR and fluorescence microscopy. This new process was proved to be efficient and stable in time up to 7 days.

X‐Ray Reflectometry Characterization of Plasma Polymer Films Synthesized from Triallylamine: Density and Swelling in Water

AbstractCommercial membranes suitable for solid alkaline membrane fuel cell (SAMFC) have not proved their competitiveness yet, due to poor ionic conductivity and water and fuel retention. Plasma polymers are however promising candidates to meet the SAMFC requirements. In this work, X‐ray reflectometry has been used to determine the thickness, density and water swelling of plasma polymerized films prepared from triallylamine, leading to a bilayer model for the studied thin films. Both film density and water swelling show a bimodal evolution as a function of the average plasma discharge power, which is directly related to the precursor fragmentation mechanism in the plasma phase and can be correlated with the film chemical composition. magnified image

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Chemical Vapor Deposition of Ni–C Films from Bis-(Ethylcyclopentadienyl) Nickel

Metal-organic chemical vapor deposition (MOCVD) is widely used for deposition of various nickel-containing coatings, such as catalytic nickel thin films and nanoparticles, nickel silicide alloys and magnetic carbon-nickel nanocomposite layers. Here we report preliminary results from an attempt to use bis-(ethylcyclopentadienyl) nickel [(EtCp)2Ni] as a precursor for MOCVD of Ni-C thin films in the (EtCp)2Ni-Ar and (EtCp)2Ni-H2-Ar reaction systems. Mechanism of precursor fragmentation was proposed on the basis of the results from a study of gaseous reaction products in the exhaust line of the reactor by means of mass-spectrometry. It was found that an introduction of hydrogen in the gas phase led to an increase in conversion rate of the precursor. Deposited films were analyzed by means of atomic force microscope (AFM), scanning electron microscopy (SEM), X-ray fluorescence spectroscopy (XFS). The effect of hydrogen on growth rate, composition, and morphology of the deposited Ni-C films were experimentally studied.

Physico‐Chemical Characterization of Methyl Isobutyrate‐based Plasma Polymer Films

AbstractMethyl isobutyrate is plasma‐polymerized varying the precursor flow rate (Φp), the working pressure (p) and the radiofrequency power injected in the discharge (PRF). Through the use of an experimental design, PRF reveals to be the key parameter regarding the incorporation of oxygen‐based functionalities in the plasma polymer films (PPFs). Combining the use of X‐ray photoelectron spectroscopy (XPS), time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and principal component analysis (PCA), our results demonstrate that an increase of PRF (i) lowers the incorporation of the precursor ester groups within the PPFs and (ii) amplifies the PPF crosslinking density (χ). A correlation between the PPF physico‐chemistry and the plasma composition is established using residual gas analysis mass spectrometry. It is shown that an increase of PRF increases both the extent of the precursor fragmentation and the production of non‐reactive oxygen‐based species in the plasma that do not contribute to the PPF growth. Incidentally, in the PRF range studied, the total amount of oxygen‐based functionalities incorporated in the PPFs (at.%O) varies from 23.9 to 10.1 at.%. magnified image

Apex Peptide Elution Chain Selection: A New Strategy for Selecting Precursors in 2D-LC−MALDI-TOF/TOF Experiments on Complex Biological Samples

LC-MALDI provides an often overlooked opportunity to exploit the separation between LC-MS and MS/MS stages of a 2D-LC-MS-based proteomics experiment, that is, by making a smarter selection for precursor fragmentation. Apex Peptide Elution Chain Selection (APECS) is a simple and powerful method for intensity-based peptide selection in a complex sample separated by 2D-LC, using a MALDI-TOF/TOF instrument. It removes the peptide redundancy present in the adjacent first-dimension (typically strong cation exchange, SCX) fractions by constructing peptide elution profiles that link the precursor ions of the same peptide across SCX fractions. Subsequently, the precursor ion most likely to fragment successfully in a given profile is selected for fragmentation analysis, selecting on precursor intensity and absence of adjacent ions that may cofragment. To make the method independent of experiment-specific tolerance criteria, we introduce the concept of the branching factor, which measures the likelihood of false clustering of precursor ions based on past experiments. By validation with a complex proteome sample of Arabidopsis thaliana, APECS identified an equivalent number of peptides as a conventional data-dependent acquisition method but with a 35% smaller work load. Consequently, reduced sample depletion allowed further selection of lower signal-to-noise ratio precursor ions, leading to a larger number of identified unique peptides.

Improving Protein and Proteome Coverage through Data-Independent Multiplexed Peptide Fragmentation

Performance differences in protein and proteome characterization achieved by data-independent acquisition (DIA) LC/MS(E) and data-dependent acquisition (DDA) LC/MS/MS approaches were investigated. LC/MS(E) is a novel mode of generating product ion data for all coeluting precursors in parallel as opposed to LC/MS/MS where coeluting precursors must be serially fragmented one at a time. During LC/MS(E) analysis, alternating MS scans of "normal" and "elevated" collision energy are collected at regular intervals, providing nearly a 100% duty cycle for precursor detection and fragmentation because all precursors are fragmented across their full chromatographic elution profile. This is in contrast to DDA-based MS/MS where serial selection of precursor ions is biased toward interrogation and detection of the highest abundance sample components by virtue of the intensity-driven interrogation scheme employed. Both modes of acquisition were applied to a simple four-protein standard mixture with a 16-fold dynamic range in concentration, an in-gel digest of the Arabidopsis thaliana protein FLS2 purified by immunoprecipitation, and a solution-digested tomato leaf proteome sample. Dramatic improvement for individual protein sequence coverage was obtained for all three samples analyzed by the DIA approach, particularly for the lowest abundance sample components. In many instances, precursors readily detected and identified during DIA were either interrogated by MS/MS during DDA at inopportune points in their chromatographic elution profiles resulting in poor quality product ion spectra or not interrogated at all. Detailed evaluation of both DDA and DIA raw data and timing of the MS-to-MS/MS switching events clearly revealed the fundamental limitations of serial MS/MS interrogation and the advantages of parallel fragmentation by DIA for more comprehensive protein identification and characterization which holds promise for enhanced isoform and post-translational modification analysis.

Deposition of Functional Organic Thin Films by Pulsed Plasma Polymerization: A Joint Theoretical and Experimental Study

AbstractThe pulsed plasma polymerization of allylamine and cyclopropylamine is investigated to study the influence of the precursor chemical structure on the process selectivity toward the primary amine groups. Both systems are compared under similar mean powers injected in the discharges (Pmean). The results reveal an increase in the precursor fragmentation in the plasma and a decrease in the primary amine content of the film (%NH2) as Pmean increases. Nevertheless, below a critical Pmean value, different behaviors are observed depending on the precursor, cyclopropylamine being less plasma‐fragmented than allylamine. As a result, plasma polymer films (PPF) synthesized from cyclopropylamine yield the largest %NH2. These results are rationalized with the help of theoretical calculations pointing to a preferential opening of the cyclopropylamine ring structure. Cyclopropylamine activation in the plasma can thus be achieved without fragmentation reactions, leading to a more efficient incorporation of the NH2 group of the precursor in the PPF.magnified image

Non-linear classification for on-the-fly fractional mass filtering and targeted precursor fragmentation in mass spectrometry experiments

Mass spectrometry (MS) has become the method of choice for protein/peptide sequence and modification analysis. The technology employs a two-step approach: ionized peptide precursor masses are detected, selected for fragmentation, and the fragment mass spectra are collected for computational analysis. Current precursor selection schemes are based on data- or information-dependent acquisition (DDA/IDA), where fragmentation mass candidates are selected by intensity and are subsequently included in a dynamic exclusion list to avoid constant refragmentation of highly abundant species. DDA/IDA methods do not exploit valuable information that is contained in the fractional mass of high-accuracy precursor mass measurements delivered by current instrumentation. We extend previous contributions that suggest that fractional mass information allows targeted fragmentation of analytes of interest. We introduce a non-linear Random Forest classification and a discrete mapping approach, which can be trained to discriminate among arbitrary fractional mass patterns for an arbitrary number of classes of analytes. These methods can be used to increase fragmentation efficiency for specific subsets of analytes or to select suitable fragmentation technologies on-the-fly. We show that theoretical generalization error estimates transfer into practical application, and that their quality depends on the accuracy of prior distribution estimate of the analyte classes. The methods are applied to two real-world proteomics datasets. All software used in this study is available from http://software.steenlab.org/fmf hanno.steen@childrens.harvard.edu Supplementary data are available at Bioinformatics online.

Rapid Validation of Mascot Search Results via Stable Isotope Labeling, Pair Picking, and Deconvolution of Fragmentation Patterns

Conventional LC-MS/MS data analysis matches each precursor ion and fragmentation pattern to their best fit within databases of theoretical spectra, yielding a peptide identification. Confidence is estimated by a score but can be validated by statistics, false discovery rates, and/or manual validation. A weakness is that each ion is evaluated independently, discarding potentially useful cross-correlations. In a classical approach to de novo sequence analysis, mixtures of peptides differing only in a carboxyl-terminal isotopic label yield fragmentation spectra with single, unlabeled b-type ions but pairs of isotope-labeled y-type ions, facilitating confident assignments. To apply this principle to identification by fragmentation pattern matching, we developed Validator, software that recognizes isotopic peptide pairs and compares their identifications and fragmentation patterns. Testing Validator 1 on a Mascot results file from FT-ICR LC-MS/MS of (16)O/(18)O-labeled yeast cell lysate peptides yielded 2,775 peptide pairs sharing a common identification but differing in carboxyl-terminal label. Comparing observed b- and y-ions with the predicted fragmentation pattern improved the threshold Mascot score for 5% false discovery from 36 to 22, significantly increasing both sensitivity and specificity. Validator 2, which identifies pairs by precursor mass difference alone before comparing observed fragmentation with that predicted by Mascot, found 2,021 isotopic pairs, similarly achieving improved sensitivity and specificity. Finally Validator 3, which finds pairs based on mass difference alone and then deconvolutes fragmentation patterns independently of Mascot, found 964 predicted peptides. Validator 3 allowed raw mass spectrometry data to be mined not only to validate Mascot results but also to discover peptides missed by Mascot. Using standard desktop hardware, the Validator 1-3 software processed the 11,536 spectra in the 93-MB Mascot .DAT file in less than 6 min (32 spectra/s), revealing high confidence peptide identifications without regard to Mascot score, far faster than manual or other independent validation methods.

CE50: Quantifying Collision Induced Dissociation Energy for Small Molecule Characterization and Identification

Survival yield analysis is routinely used in mass spectroscopy as a tool for assessing precursor ion stability and internal energy. Because ion internal energy and decomposition reaction rates are dependent on chemical structure, we reasoned that survival yield curves should be compound-specific and therefore useful for chemical identification. In this study, a quantitative approach for analyzing the correlation between survival yield and collision energy was developed and validated. This method is based on determining the collision energy (CE) at which the survival yield is 50% (CE(50)) and, further, provides slope and intercept values for each survival yield curve. In initial experiments using a defined set of homologous compounds, we found that CE(50) values were easily determined, quantitative, highly reproducible, and could discriminate between structural and even positional isomers. Further analysis demonstrated that CE(50) values were independent of cone potential and orthogonal to compound mass. Experimentally determined CE(50) values for a diverse set of 54 compounds were correlated to Molconn molecular structure descriptors. The resulting model yielded a statistically significant linear correlation between experimental and calculated CE(50) values and identified several structural characteristics related to precursor ion stability and fragmentation mechanism. Thus, the CE(50) is a promising method for compound identification and discrimination.

Synthesis of Allylamine Plasma Polymer Films: Correlation between Plasma Diagnostic and Film Characteristics

AbstractPrimary amine‐based plasma polymer films (NH2‐PPF) were synthesized using plasma polymerization of allylamine in continuous wave (CW) and pulsed radio‐frequency (RF) modes. Plasma chemistry, studied by residual gas analysis mass spectrometry, revealed that the precursor fragmentation is a function of the equivalent power (Peq) dissipated in the discharge, independently of the plasma mode used. X‐ray photoelectron spectroscopy combined with time‐of‐flight secondary ion mass spectrometry suggests as the precursor fragmentation in the plasma increases: (i) a decrease of the primary amine concentration in the NH2‐PPF (%NH2) and (ii) an increase of the cross‐linking degree. For a given Peq, similar to the precursor fragmentation in the plasma, the NH2‐PPF characteristics were found to be independent of the plasma mode used. Therefore, the main advantage of using pulsed RF processes over CW ones is the possibility to work at very low Peq which enables low precursor fragmentation, optimization of %NH2, and reduction of the film cross‐linking degree. The chemical composition and the cross‐linking degree of the NH2‐PPF synthesized by allylamine plasma polymerization can thus be tailored by adjusting the equivalent RF power injected in the plasma.magnified image