LTQ Velos Research Articles

Egg perivitelline fluid proteome of a freshwater snail: Insight into the transition from aquatic to terrestrial egg deposition.

Proteins from the egg perivitelline fluid (PVF) are assumed to play critical roles in embryonic development, but for many groups of animals their identities remain unknown. Identifying egg PVF proteins is a critical step towards understanding their functions including their roles in evolutionary transition in habitats. We applied proteomic and transcriptomic analysis to investigate the PVF proteome of the eggs of Pomacea diffusa, an aerial ovipositing freshwater snail in the family Ampullariidae. The PVF proteins were separated with the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method, and proteomic analysis was conducted using an LTQ Velos ion trap mass spectrometer coupled with liquid chromatography. Comparison of PVF proteomes and evolution analyses was performed between P. diffusa and other ampullariids. In total, 32 egg PVF proteins were identified from P. diffusa. They were categorized as PV1-like subunits, immune-responsive proteins, protein degradation, signaling and binding, transcription and translation, metabolism, oxidation-reduction and proteins with unknown function. Interestingly, the proteome includes a calcium-binding protein important in forming the hard eggshell that enabled the terrestrial transition. However, it does not include PV2, a neurotoxic protein that was assumed to be present in all Pomacea species. The PVF proteome data from P. diffusa can help us better understand the roles that reproductive proteins played during the transition from underwater to terrestrial egg deposition. Moreover, they could be useful in comparative studies of the terrestrialization in several groups of animals that occurred independently during their evolution.

Liquid chromatography – high resolution mass spectrometry method for monitoring of 17 mycotoxins in human plasma for exposure studies

Mycotoxins are secondary metabolites produced by filamentous fungi. Primary route of human exposure to mycotoxins is the intake of the contaminated food. Minimizing mycotoxin exposure is important for population health, as their chronic toxic effects have been associated with kidney and liver diseases, some types of cancer and immunosuppression. The objective of this work was to develop and validate a multi-class mycotoxin method suitable for exposure monitoring of mycotoxins in human plasma. A sensitive liquid chromatography – mass spectrometry method was developed for 17 mycotoxins: nivalenol (NIV), deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, T-2 toxin, HT-2 toxin, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, zearalenone, α-zearalenol (α-ZOL), β-zearalenol, zearalanone, α-zeranol and, and β-zeranol. The method relies on three-step liquid–liquid extraction with ethyl acetate to eliminate the need for immunoaffinity extraction and minimize ionization matrix effects. Chromatographic separation of mycotoxins, including all isomers, was achieved with pentafluorophenyl column and water/methanol mobile phase. Mycotoxin detection and quantitation were performed using high-resolution mass spectrometry on LTQ Velos Orbitrap, in both positive and negative electrospray ionization (ESI(+) and (ESI(−)). The use of 0.02% acetic acid as mobile phase additive for ESI(−) resulted in significant increase in ionization efficiency ranging from 1.7 to 26 times for mycotoxins that ionize better in ESI(−). The optimized method was validated according to FDA guidance procedures. LOQs of all mycotoxins ranged from 0.1 to 0.5 ng/ml, except NIV which resulted in LOQ of 3 ng/ml because of low extraction recovery of this highly polar mycotoxin. Mean intra-day accuracy ranged from 85.8% to 116.4%, and intra-day precision (n = 6) ranged from 1.6% to 12.5% RSD for all mycotoxins except α-ZOL where mean accuracy ranged from 72.9% to 97.2%. Inter-day accuracy and precision were 85.6%–111.5% and 2.7–15.6% RSD respectively, showing good analytical performance of the method for biomonitoring.

Cervical Alpha-Actinin-4 Is Upregulated in Women with Threatened Preterm Labor and Microbial Invasion of the Amniotic Cavity

Objective: To characterize the proteome profile of women with threatened preterm labor (PTL) below 34;0 weeks with and without microbial invasion of the amniotic cavity (MIAC) using mass spectrometry in the amniotic fluid (AF) and Western blot analysis in the cervical mucus and the vaginal fluid. Subjects and Methods: In the discovery phase, a case-control study including 8 women with MIAC and 7 without matched for gestational age at sampling was performed. Proteomic profile characterization was done using the LTQ VELOS Orbitrap mass spectrometer in the AF. In the validation phase, a selection of the proteins differentially expressed by mass spectrometry in the genital samples of a prospective cohort of 109 women was validated by Western blot analysis. Results: In the discovery phase, the mass spectrometry analysis identified a total of 444 proteins. Sixteen were chosen for validation, being involved in defense (calgranulin A, B, C, C-reactive protein), cytoskeletal remodeling (alpha-actinin-4 [ACTN-4], plastin-2, α2-antiplasmin, vitronectin), metabolism (cystatin-β, glucose 6 phosphate isomerase, glutathione S-transferase, prostaglandin D2 synthase, corticosteroid-binding globulin), and vascular (α1-antichymotrypsin, hemopexin, endosialin) pathways. In the validation phase, cervical ACTN-4 was the only significantly upregulated protein in women with MIAC with an odds ratio of 6.8 (p = 0.002). Conclusions: Cervical ACTN-4 was significantly upregulated in the group of women with PTL with MIAC.

Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques

One important challenge in lipid class analysis is to develop a method suitable or, at least adaptable, for a vast diversity of samples. In the current study, an improved normal-phase liquid chromatography (NPLC) method allowed analyzing the lipid classes present in mammalian, vegetable as well as microorganism (yeast and bacteria) lipid samples. The method effectively separated 30 lipid classes or subclasses with a special focus on medium polarity lipids. The separation was carried out with bare silica stationary phase and was coupled to evaporative light scattering detection (ELSD), charged aerosol detection (Corona-CAD®) and mass spectrometry. Solutions are provided to circumvent technical issues (such as pumping solvents of low viscosity, solvent purity, rinsing step). The influence of mobile phase composition and addition of ionic modifiers on the chromatographic behavior of particular lipid classes is documented. A comparison between ELSD and Corona-CAD® confirmed the interest of this later detector for samples with a wide range of concentration of different lipids. Three common atmospheric pressure ionization interfaces were used for coupling the NPLC separation to a LTQ Velos Pro® mass spectrometer. The comparison of the chromatographic profiles showed that atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) are both suitable to detect the different lipid classes whereas APPI allows a better sensitivity for lipids at low-concentration.

Proteomic analysis of the kissing bug Rhodnius prolixus antenna

Reception of odorants is essential in insects’ life since the chemical signals in the environment (=semiochemicals) convey information about availability of hosts for a blood meal, mates for reproduction, sites for oviposition and other relevant information for fitness in the environment. Once they reach the antennae, these semiochemicals bind to odorant-binding proteins and are transported through the sensillar lymph until reach the odorant receptors. Such perireceptor events, particularly the interactions with transport proteins, are the liaison between the external environment and the entire neuroethological system and, therefore, a potential target to disrupt insect chemical communication. In this study, a proteomic profile of female and male antennae of Rhodnius prolixus, a vector of Chagas disease, was obtained in an attempt to unravel the entire repertoire of olfactory proteins involved in perireceptor events. Using shotgun proteomics and two-dimensional gel electrophoresis approaches followed by nano liquid chromatography coupled with tandem LTQ Velos Orbitrap mass spectrometry, we have identified 581 unique proteins. Putative olfactory proteins, including 17 odorant binding proteins, 6 chemosensory proteins, 2 odorant receptors, 3 transient receptor channels and 1 gustatory receptor were identified. Proteins involved in general cellular functions such as generation of precursor metabolites, energy generation and catabolism were expressed at high levels. Additionally, proteins that take part in signal transduction, ion binding, and stress response, kinase and oxidoreductase activity were frequent in antennae from both sexes. This proteome strategy unraveled for the first time the complex nature of perireceptor and other olfactory events that occur in R. prolixus antennae, including evidence for phosphorylation of odorant-binding and chemosensory proteins. These findings not only increase our understanding of the olfactory process in triatomine species, but also identify potential molecular targets to be explored for population control of such insect vectors.

Dissociation of doubly charged clusters of lithium acetate: Asymmetric fission and breakdown of the liquid drop model

Electrospray ionization (ESI) results in the formation of multiply charged cluster ions which then fragment via two competing processes: fission (charge separation) via Coulomb repulsion, and evaporation of one or more neutrals from the fragmenting ion, as described by Rayleigh's liquid drop model. Unimolecular and collision-induced dissociation (CID) of doubly charged lithium acetate clusters, (CH3 COOLi)n Li2 (2+) , demonstrated that Coulomb fission via charge separation is the dominant dissociation process with no contribution from the neutral evaporation processes for all such ions from the critical limit to larger cluster ions, although the latter process was normally observed in most earlier studies of multiply charged clusters. Doubly and higher charged lithium acetate cluster ions were generated by ESI. Unimolecular and CID of mass selected doubly charged cluster ions above the critical limit were recorded at high resolution using an LTQ Velos Orbitrap mass spectrometer. Unimolecular and collision-induced dissociation of doubly charged lithium acetate clusters proceed via symmetric fission processes and the intensity of the fragment ions decreases as the cluster size increases. Dissociations via neutral evaporation of a monomer or larger clusters are not observed. Doubly charged clusters of lithium acetate above the critical limit dissociate via symmetrical fission processes and not neutral evaporation. These results are clearly in disagreement with the Rayleigh liquid drop model that has been used previously to predict the critical size and explain the fragmentation behavior of multiply charged clusters. Copyright © 2016 John Wiley & Sons, Ltd.

DNA Interactions Probed by Hydrogen-Deuterium Exchange (HDX) Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Confirm External Binding Sites on the Minichromosomal Maintenance (MCM) Helicase

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect single-stranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5-30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.

Portable, Battery Operated Capillary Electrophoresis with Optical Isomer Resolution Integrated with Ionization Source for Mass Spectrometry.

We introduce a battery operated capillary electrophoresis electrospray ionization (CE/ESI) source for mass spectrometry with optical isomer separation capability. The source fits in front of low or high resolution mass spectrometers similar to a nanospray source with about the same weight and size. The source has two high voltage power supplies (±25 kV HVPS) capable of operating in forward or reverse polarity modes and powered by a 12 V rechargeable lithium ion battery with operation time of ~10 h. In ultrafast CE mode, in which short narrow capillaries (≤15 μm i.d., 15-25 cm long) and field gradients ≥1000 V/cm are used, peak widths at the base are <1 s wide. Under these conditions, the source provides high resolution separation, including optical isomer resolution in ~1 min. Using a low resolution mass spectrometer (LTQ Velos) with a scan time of 0.07 s/scan, baseline separation of amino acids and their optical isomers were achieved in ~1 min. Moreover, bovine serum albumin (BSA) was analyzed in ~1 min with 56% coverage using the data-dependent MS/MS. Using a high resolution mass spectrometer (Thermo Orbitrap Elite) with 15,000 resolution, the fastest scan time achieved was 0.15 s, which was adequate for CE-MS analysis when optical isomer separation is not required or when the optical isomers were well separated. Figures of merit including a detection limit of 2 fmol and linear dynamic range of two orders of magnitude were achieved for amino acids.

Reproducibility and quantification of illicit drugs using matrix-assisted ionization (MAI) mass spectrometry.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

In-line separation by capillary electrophoresis prior to analysis by top-down mass spectrometry enables sensitive characterization of protein complexes.

Intact protein analysis via top-down mass spectrometry (MS) provides a bird’s eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE–electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment.

High resolution is not a strict requirement for characterization and quantification of histone post-translational modifications.

Mass spectrometry (MS) is a powerful tool to accurately identify and quantify histone post-translational modifications (PTMs). High-resolution mass analyzers have been regarded as essential for these PTM analyses because the mass accuracy afforded is sufficient to differentiate trimethylation versus acetylation (42.0470 and 42.0106 Da, respectively), whereas lower-resolution mass analyzers cannot. Noting this limitation, we sought to determine whether lower-resolution detectors are nonetheless adequate for histone PTM analysis by comparing the low-resolution LTQ Velos Pro with the high-resolution LTQ-Orbitrap Velos Pro. We first determined that the optimal scan mode on the LTQ Velos Pro is the Enhanced scan mode with respect to apparent resolution, number of MS and MS/MS scans per run, and reproducibility of label-free quantifications. We next compared the performance of the LTQ Velos Pro to the LTQ-Orbitrap Velos Pro using the same criteria for comparison, and we found that the main difference is that the LTQ-Orbitrap Velos Pro is able to resolve the difference between acetylation and trimethylation while the LTQ Velos Pro cannot. However, using heavy isotope labeled synthetic peptide standards and retention time information enables confident assignment of these modifications and comparable quantification between the instruments. Therefore, lower-resolution instruments can confidently be utilized for histone PTM analysis.

Comparison of data acquisition strategies on quadrupole ion trap instrumentation for shotgun proteomics.

The most common data collection in shotgun proteomics is via data-dependent acquisition (DDA), a process driven by an automated instrument control routine that directs MS/MS acquisition from the highest abundant signals to the lowest. An alternative to DDA is data-independent acquisition (DIA), a process in which a specified range in m/z is fragmented without regard to prioritization of a precursor ion or its relative abundance in the mass spectrum, thus potentially offering a more comprehensive analysis of peptides than DDA. In this work, we evaluate both DDA and DIA on three different linear ion trap instruments: an LTQ, an LTQ modified with an electrodynamic ion funnel, and an LTQ Velos. These instruments represent both older (LTQ) and newer (LTQ Velos) ion trap designs (i.e., linear versus dual ion traps, respectively), and allow direct comparison of peptide identifications using both DDA and DIA analysis. Further, as the LTQ Velos has an enhanced "S-lens" ion guide to improve ion flux, we found it logical to determine if the former LTQ model could be leveraged by improving sensitivity by modifying with an electrodynamic ion guide of significantly different design to the S-lens. We find that the ion funnel enabled LTQ identifies more proteins in the insoluble fraction of a yeast lysate than the other two instruments in DIA mode, whereas the faster scanning LTQ Velos performs better in DDA mode. We explore reasons for these results, including differences in scan speed, source ion optics, and linear ion trap design.

Statistical characterization of HCD fragmentation patterns of tryptic peptides on an LTQ Orbitrap Velos mass spectrometer.

Orbitrap mass spectrometry is becoming the popular instrument for proteomic analysis, and HCD mode is becoming the main analysis mode for its high resolution and high sensitivity. This study characterizes the features of HCD spectrum by comparing with CID mode on LTQ Velos Orbitrap. The patterns of b and y ions in HCD and CID modes were systematically compared for the first time, including the charge state, ion frequency and intensity and cleavage selectivity. These results will help develop database search engines and design proper transitions for targeted proteomic analysis in the future.

Characterizing synthetic polymers and additives using new ionization methods for mass spectrometry.

New inlet and vacuum ionization methods provide advantages of specificity, simplicity and speed for the analysis of synthetic polymers and polymer additives directly from surfaces such as fibers using mass spectrometry (MS) on different commercial mass spectrometers (Waters SYNAPT G2, Thermo LTQ Velos). We compare inlet ionization methods with the recently discovered vacuum ionization method. This method, termed matrix assisted ionization vacuum (MAIV), utilizes the matrix 3-nitrobenzonitrile (3-NBN) for the analysis of synthetic polymers and additives without additional energy input by simply exposing the matrix:analyte:salt to the vacuum of the mass spectrometer. Matrix:analyte:salt samples can be introduced while dry (surfaces, e.g. glass slides, pipet tips) or slightly wet (e.g. filter paper, pipet tips). Compounds ionized by these methods can be analyzed in both positive and negative detection modes through cationization or deprotonation, respectively. The dynamic range of the experiment can be enhanced, as well as structural analysis performed, by coupling the vacuum ionization method with ion mobility spectrometry mass spectrometry (IMS-MS) and tandem mass spectrometric (MS/MS) fragmentation. The specificity of 3-NBN matrix to ionize small and large nonvolatile analyte molecules by MAIV makes this matrix a good choice for observing low-abundance additives in the presence of large amounts of synthetic polymer using MS.

Relative quantification of the effect of blood meal iron on protein expression in the ovaries of Aedes aegypti (778.8)

Diseases transmitted by mosquitoes are estimated to kill more than 2 million people annually. The vector for dengue, Aedes aegypti (yellow fever mosquito), is found in the desert Southwest of the continental United States. One approach to limiting the spread of disease is to reduce the numbers of mosquito vectors. Our area of research is iron metabolism. Our long term goal is to use molecular techniques to alter the expression of iron related proteins in mosquitoes to reduce mosquito fecundity and survival. Our hypothesis is that female mosquitoes require iron in the blood meal as an essential nutrient to aid in egg development. Although the iron is necessary for fecundity it is also highly oxidative and toxic in biological systems unless the mineral is strictly regulated to mitigate its oxidative potential. We are currently analyzing tissue‐specific, iron regulated proteins of the mosquito ovary 24 h post‐blood meal fed on artificial blood meal diets supplemented with no iron or low iron, or a porcine blood meal (high iron). Measurement of the blood meal iron effect on ovarian protein expression was carried out using Tandem Mass Tags (TMT) which permits simultaneous identification and relative quantification of proteins during LTQ Velos Orbitrap LC‐MS/MS analysis. This research will contribute to our understanding of mosquito development in general, as well as ovarian iron metabolism.Grant Funding Source: Supported by funds from the Arizona Ag Experiment Station and CALS

Accurate Label-Free Protein Quantitation with High- and Low-Resolution Mass Spectrometers

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well-correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10(-3) or lower were detected, with a strong falloff below 10(-4) mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate but should be limited to quantitation of proteins of intermediate to higher fractional abundance.

N- and O-Glycosylation in the Murine Synaptosome

We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.

Putting the Pieces Together: High-performance LC-MS/MS Provides Network-, Pathway-, and Protein-level Perspectives in Populus

High-performance mass spectrometry (MS)-based proteomics enabled the construction of a detailed proteome atlas for Populus, a woody perennial plant model organism. Optimization of experimental procedures and implementation of current state-of-the-art instrumentation afforded the most detailed look into the predicted proteome space of Populus, offering varying proteome perspectives: (1) network-wide, (2) pathway-specific, and (3) protein-level viewpoints. Together, enhanced protein retrieval through a detergent-based lysis approach and maximized peptide sampling via the dual-pressure linear ion trap mass spectrometer (LTQ Velos), have resulted in the identification of 63,056 tryptic peptides. The technological advancements, specifically spectral-acquisition and sequencing speed, afforded the deepest look into the Populus proteome, with peptide abundances spanning 6 orders of magnitude and mapping to ∼25% of the predicted proteome space. In total, tryptic peptides mapped to 11,689 protein assignments across four organ-types: mature (fully expanded, leaf plastichronic index (LPI) 10-12) leaf, young (juvenile, LPI 4-6) leaf, root, and stem. To resolve protein ambiguity, identified proteins were grouped by sequence similarity (≥ 90%), thereby reducing the protein assignments into 7538 protein groups. In addition, this large-scale data set features the first systems-wide survey of protein expression across different Populus organs. As a demonstration of the precision and comprehensiveness of the semiquantitative analysis, we were able to contrast two stages of leaf development, mature versus young leaf. Statistical comparison through ANOVA analysis revealed 1432 protein groups that exhibited statistically significant (p ≤ 0.01) differences in protein abundance. Experimental validation of the metabolic circuitry expected in mature leaf (characterized by photosynthesis and carbon fixation) compared with young leaf (characterized by rapid growth and moderate photosynthetic activities) strongly testifies to the credibility of the approach. Instead of quantitatively comparing a few proteins, a systems view of all the changes associated with a given cellular perturbation could be made.