Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface.

Patients with cystic fibrosis (CF) have decreased severity of severe acute respiratory syndrome-like coronavirus-2 (SARS-CoV-2) infections, but the underlying cause is unknown. Patients with CF have high levels of neutrophil elastase (NE) in the airway. We examined whether respiratory epithelial angiotensin-converting enzyme 2 (ACE-2), the receptor for the SARS-CoV-2 spike protein, is a proteolytic target of NE. Soluble ACE-2 levels were quantified by ELISA in airway secretions and serum from patients with and without CF, the association between soluble ACE-2 and NE activity levels was evaluated in CF sputum. We determined that NE activity was directly correlated with increased ACE-2 in CF sputum. Additionally, primary human bronchial epithelial (HBE) cells, exposed to NE or control vehicle, were evaluated by Western analysis for the release of cleaved ACE-2 ectodomain fragment into conditioned media, flow cytometry for the loss of cell surface ACE-2, its impact on SARS-CoV-2 spike protein binding. We found that NE treatment released ACE-2 ectodomain fragment from HBE and decreased spike protein binding to HBE. Furthermore, we performed NE treatment of recombinant ACE-2-Fc-tagged protein in vitro to assess whether NE was sufficient to cleave recombinant ACE-2-Fc protein. Proteomic analysis identified specific NE cleavage sites in the ACE-2 ectodomain that would result in loss of the putative N-terminal spike-binding domain. Collectively, data support that NE plays a disruptive role in SARS-CoV-2 infection by catalyzing ACE-2 ectodomain shedding from the airway epithelia. This mechanism may reduce SARS-CoV-2 virus binding to respiratory epithelial cells and decrease the severity of COVID19 infection.

Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

Ultraviolet photodissociation of fondaparinux generates signature antithrombin-like 3-O-sulfated -GlcNS3S6S- monosaccharide fragment (Y3/C3).

The 3-O sulfate-modified -GlcNS3S6S- monosaccharide in heparin and heparan sulfate glycosaminoglycans (HSGAGs) is a relatively rare yet important modification that facilitates HSGAG-antithrombin binding and subsequent anticoagulant activity. Detecting this modification in complex HSGAG mixtures is a longstanding goal to identify novel 3-O-sulfated HSGAG-protein interactions with biologically significant functions. Tandem mass spectrometry has been applied to HSGAG structural analysis but is limited by the fact that traditional collision-induced dissociation techniques (e.g., CID, HCD) results in extensive sulfate loss prior to generating structurally informative glycosidic and cross-ring fragments. In the present study, we investigated the potential of ultraviolet photodissociation (UVPD) to generate structurally informative fragments from the synthetic heparin mimetic, fondaparinux, under electrospray conditions commensurate with hydrophilic interaction liquid chromatography (HILIC). The two predominant un-adducted precursors, [Fonda-2H+]2- and [Fonda-3H+]3-, were subjected to UVPD, CID, and HCD on an Orbitrap Fusion Lumos Tribrid mass spectrometer and the resulting fragmentation spectra directly compared. Close inspection of the UVPD data identified a unique peak at m/z 417.9425 that matched the Y3/C3 double glycosidic fragment of fondaparinux (i.e., -GlcNS3S6S-). Importantly, the 3-O-sulfated Y3/C3 fragment was generated predominantly from UVPD of the [Fonda-2H+]2- precursor, increased with activation time, and was observable using data-dependent HILIC-MS/MS UVPD analysis of fondaparinux spiked into a semi-complex HSGAG mixture. The discovery of this antithrombin-like 3-O-sulfated fragment provides a potential strategy for screening complex HSGAG mixtures in a data-dependent or data-independent acquisition mode to determine the presence of this therapeutic and biologically significant HSGAG modification. Graphical abstract.

The lignan manassantin is a potent and specific inhibitor of mitochondrial complex I and bioenergetic activity in mammals.

Dineolignans manassantin A and B from the plant Saururus cernuus are used in traditional medicine to manage a wide range of ailments such as edema, jaundice, and gonorrhea. Cell-based studies have identified several molecular target candidates of manassantin including NF-κB, MAPK, STAT3, and hypoxia-inducible factor 1α (HIF-1α). It is unclear whether or how these structurally diverse proteins or pathways mediate any of the medical benefits of manassantin in vivo Moreover, it has recently been reported that manassantin causes developmental arrest in zebrafish by inhibiting the mitochondrial complex I, but it is unknown whether manassantin inhibits mitochondrial respiration in intact mammalian cells and live animals. Here, we present direct evidence that manassantin potently and specifically inhibits the mitochondrial complex I and bioenergetic activity in mammalian systems. Manassantin had no effect on complex II- or complex IV-mediated respiration. Of note, it decreased NADH-ubiquinone reductase activity but not the activity of NADH-ferricyanide reductase. Treatment with manassantin reduced cellular ATP levels and concomitantly stimulated AMP-activated protein kinase in vitro and in vivo As an adaptive response to manassantin-induced bioenergetic deficiency, mammalian cells up-regulated aerobic glycolysis, a process mediated by AMP-activated protein kinase (AMPK) independently of HIF-1α. Together these results demonstrate a biologically important activity of manassantin in the control of complex I-mediated respiration and its profound effects on oxygen utilization, energy homeostasis, and glucose metabolism in mammalian cells.

Proteomics Insights into Autophagy.

Autophagy, a conserved cellular process by which cells recycle their contents either to maintain basal homeostasis or in response to external stimuli, has for the past two decades become one of the most studied physiological processes in cell biology. The 2016 Nobel Prize in Medicine and Biology awarded to Dr. Ohsumi Yoshinori, one of the first scientists to characterize this cellular mechanism, attests to its importance. The induction and consequent completion of the process of autophagy results in wide ranging changes to the cellular proteome as well as the secretome. MS-based proteomics affords the ability to measure, in an unbiased manner, the ubiquitous changes that occur when autophagy is initiated and progresses in the cell. The continuous improvements and advances in mass spectrometers, especially relating to ionization sources and detectors, coupled with advances in proteomics experimental design, has made it possible to study autophagy, among other process, in great detail. Innovative labeling strategies and protein separation techniques as well as complementary methods including immuno-capture/blotting/staining have been used in proteomics studies to provide more specific protein identification. In this review, we will discuss recent advances in proteomics studies focused on autophagy.

Comparative analysis of INLIGHT™-labeled enzymatically depolymerized heparin by reverse-phase chromatography and high-performance mass spectrometry.

Structural characterization of the microheterogeneity of heparin, heparan sulfate, and other glycosaminoglycans is a major analytical challenge. We present the use of a stable isotope-labeled hydrazide tag (INLIGHT™) with high-resolution/accurate mass (HRAM) reverse-phase LC-MS/MS, which was recently introduced for detailed study of N-glycan heterogeneity, to characterize heparinase-digested heparin (digHep) products without the use of semi-volatile ion pairing reagents. Using both full scan LC-MS and data-dependent LC-MS/MS, we identified 116 unique digHep species, a feat possible because of INLIGHT™ labeling. Of these, 83 digHep products were structurally identified, including the 12 standard disaccharides as well as 34 tetra- (DP4), 26 hexa- (DP6), 21 octa- (DP8), and 2 decasaccharides (DP10). Each of the 116 digHep species co-eluted with both light and heavy INLIGHT™ tags (L/Havg = 1.039 ± 0.163); thus enhancing confidence in their identification via MS and MS/MS. This work sets the foundation for INLIGHT™-based comparative analyses of different forms of heparin, heparan sulfate, and other GAGs with high quantitative precision using mainstay reverse-phase HRAM LC-MS/MS. Graphical Abstract Reducing end labeling strategy for mapping depolymerized heparin/heparan sulfate products by reverse-phase LC-MS/MS.

Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.

Multi-peptide nLC-PC-IDMS-SRM-based assay for the quantification of biomarkers in the chicken ovarian cancer model.

A novel form of ovomacroglobulin/ovostatin (OVOS2) predicted from EST data was previously identified in the chicken ovarian cancer model using a mass spectrometry-based shotgun label-free proteomics strategy. The quantitative label-free data from plasma showed a significant increase over time with the spontaneous onset and progression of ovarian cancer making it a potential protein biomarker for further study. Two other proteins of interest identified from this initial study included vitellogenin-1 (Vit-1), a lipid-transport protein tied to egg production, and transthyretin (TTR), a retinol binding transport protein currently used in the clinical management of ovarian cancer. A multiplexed protein cleavage isotope dilution mass spectrometry (PC-IDMS) assay was developed to quantify OVOS2, Vit-1, and TTR by selected reaction monitoring (SRM). A total of 6 stable isotope labeled (SIL) peptide standards were used in the assay with three tryptic peptides from OVOS2, one for Vit-1, and two for TTR. The assay was developed for use with un-depleted raw plasma combined with the filter assisted sample preparation (FASP) method and its use was also demonstrated for matched ovary tissue samples. The PC-IDMS data for the two TTR peptides did not correlate with each other with more than a 10-fold difference in concentration for all 5 time points measured. The PC-IDMS data from the longitudinal plasma samples correlated well for OVOS2 and Vit-1 whereas TTR was inconclusive. Interestingly, the absolute amount for one of the OVOS2 SIL peptides was 2-fold less compared with the other two SIL peptides. These data illustrate the successes and challenges of qualifying quantitative levels of proteins from an in-gel digestion sample preparation followed by LC-MS/MS (GeLC) label-free discovery-based approach to a targeted SRM-based quantitative assay in plasma and tissues.

Performance characteristics of a new hybrid quadrupole time-of-flight tandem mass spectrometer (TripleTOF 5600).

The TripleTOF 5600 System, a hybrid quadrupole time-of-flight mass spectrometer, was evaluated to explore the key figures of merit in generating peptide and protein identifications that included spectral acquisition rates, data quality, proteome coverage, and biological depth. Employing a Saccharomyces cerevisiae tryptic digest, careful consideration of several performance features demonstrated that the speed of the TripleTOF contributed most to the resultant data. The TripleTOF system was operated with 8, 20, and 50 MS/MS events in an effort to compare with other MS technologies and to demonstrate the abilities of the instrument platform.

One-Year Plasma N-linked Glycome Intra-individual and Inter-individual Variability in the Chicken Model of Spontaneous Ovarian Adenocarcinoma.

Spontaneous epithelial ovarian cancer (EOC) in the chicken presents a similar pathogenesis compared with humans including CA-125 expression and genetic mutational frequencies (e.g., p53). The high prevalence of spontaneous EOC chickens also provides a unique experimental model for biomarker discovery at the genomic, proteomic, glycomic, and metabolomic level. In an effort to exploit this unique model for biomarker discovery, longitudinal plasma samples were collected from chickens at three month intervals for one year. The study described herein involved cleaving the N-glycans from these longitudinal chicken plasma samples and analyzing them via nanoLC-FTMS/MS. Glycans identified in this study were previously found in human plasma and this work provides a promising methodology to enable longitudinal studies of the N-linked plasma glycome profile during EOC progression. The structure, abundance, and intra-variability and inter-variability for 35 N-linked glycans identified in this study are reported. The full potential of the chicken model for biomarker discovery has yet to be realized, but the initial interrogation of longitudinally-procured samples provides evidence that supports the value of this strategy in the search for glycomic biomarkers.

Proteomics characterization of cell membrane blebs in human retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation, and in-gel trypsin-digested peptides were analyzed by LC-MS/MS that lead to the identification of a total of 314 proteins. Identified proteins were predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease and shed light on the implication of cell-derived microvesicles in human pathology.

Utilizing spectral counting to quantitatively characterize tandem removal of abundant proteins (TRAP) in human plasma.

Biomarker discovery efforts in serum and plasma are greatly hindered by the presence of high abundance proteins that prevent the detection and quantification of less abundant, yet biologically significant, proteins. The most common method for addressing this problem is to specifically remove the few abundant proteins through immunoaffinity depletion/subtraction. Herein, we improved upon this method by utilizing multiple depletion columns in series, so as to increase the efficiency of the abundant protein removal and augment the detection/identification of less abundant plasma proteins. Spectral counting was utilized to make quantitative comparisons between undepleted plasma, plasma depleted with a single depletion column, and plasma depleted using two or three depletion columns in tandem. In the undepleted plasma only 29 lower abundance protein groups were identified with the top-scoring protein from each group having a median spectral count of 3, while in the plasma processed using a single HSA depletion column 61 such protein groups were identified with a median spectral count of 8. In comparison, 76 lesser abundant protein groups were identified with a median spectral count of 11.5 in the two column setup (i.e., HSA followed by MARS Hu14). However, in the ultimate depleted plasma sample, which was created using three depletion columns in tandem, the number of less abundant protein groups identified increase to 81 and the median spectral count for the top-scoring proteins from each group increased to 15 counts per protein. Moreover, exogenous B-type natriuretic peptide-32, which was added to the plasma as a detection benchmark at 12 µg/mL, was only detected in the plasma sample depleted using three depletion columns in tandem. Collectively, these data demonstrate that this method, tandem removal of abundant proteins or TRAP, provides superior removal efficiency compared to traditional applications and improves the depth of proteome coverage in plasma.

Measuring the intra-individual variability of the plasma proteome in the chicken model of spontaneous ovarian adenocarcinoma.

The domestic chicken (Gallus domesticus) has emerged as a powerful experimental model for studying the onset and progression of spontaneous epithelial ovarian cancer (EOC) with a disease prevalence that can exceed 35% between 2 and 7 years of age. An experimental strategy for biomarker discovery is reported herein that combines the chicken model of EOC, longitudinal plasma sample collection with matched tissues, advanced mass spectrometry-based proteomics, and concepts derived from the index of individuality (Harris, Clin Chem 20: 1535-1542, 1974). Blood was drawn from 148 age-matched chickens starting at 2.5 years of age every 3 months for 1 year. At the conclusion of the 1 year sample collection period, the 73 birds that remained alive were euthanized, necropsied, and tissues were collected. Pathological assessment of resected tissues from these 73 birds confirmed that five birds (6.8%) developed EOC. A proteomics workflow including in-gel digestion, nanoLC coupled to high-performance mass spectrometry, and label-free (spectral counting) quantification was used to measure the biological intra-individual variability (CV(W)) of the chicken plasma proteome. Longitudinal plasma sample sets from two birds within the 73-bird biorepository were selected for this study; one bird was considered "healthy" and the second bird developed late-stage EOC. A total of 116 proteins from un-depleted plasma were identified with 80 proteins shared among all sample sets. Analytical variability (CV(A)) of the label-free proteomics workflow was measured using a single plasma sample analyzed five times and was found to be ≥CV(W) in both birds for 16 proteins (20%) and in either bird for 25 proteins (31%). Ovomacroglobulin (ovostatin) was found to increase (p < 0.001) over a 6 month period in the late-stage EOC bird providing an initial candidate protein for further investigation.

Temporal map of the pig polytrauma plasma proteome with fluid resuscitation and intravenous vitamin C treatment.

BACKGROUND: Fluid resuscitation plays a prominent role in stabilizing trauma patients with hemorrhagic shock yet there remains uncertainty with regard to optimal administration time, volume, and fluid composition (e.g., whole blood, component, colloids) leading to complications such as trauma-induced coagulopathies (TIC), acidosis, and poor oxygen transport. Synthetic fluids in combination with antioxidants (e.g., vitamin C) may resolve some of these problems. OBJECTIVES: We applied quantitative mass spectrometry-based proteomics [liquid chromatography-mass spectrometry (LC-MS/MS)] to map the effects of fluid resuscitation and intravenous vitamin C (VitC) in a pig model of polytrauma (hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture). The goal was to determine the effects of VitC on plasma protein expression, with respect to changes associated with coagulation and trauma-induced coagulopathy (TIC). METHODS: Longitudinal blood samples were drawn from nine male Sinclair pigs at baseline, 2 h post trauma, and 0.25, 2, and 4 h post fluid resuscitation with 500 mL hydroxyethyl starch. Pigs were treated intravenously (N = 3/treatment group) with saline, 50 mg VitC/kg (Lo-VitC), or 200 mg VitC/kg (Hi-VitC) during fluid resuscitation. RESULTS: A total of 436 plasma proteins were quantified of which 136 changed following trauma and resuscitation; 34 were associated with coagulation, complement cascade, and glycolysis. Unexpectedly, Lo-VitC and Hi-VitC treatments stabilized ADAMTS13 levels by ~4-fold (P = .056) relative to saline and enhanced ADAMTS13/von Willebrand factor (VWF) cleavage efficiency based on LC-MS/MS evidence for the semitryptic VWF cleavage product (VWF1275-1286 ). CONCLUSIONS: This study provides the first comprehensive map of trauma-induced changes to the plasma proteome, especially with respect to proteins driving the development of TIC.

Coordination-driven face-directed self-assembly of trigonal prisms. Face-based conformational chirality.

The coordination-driven self-assembly of four different trigonal prisms from 3 equiv of one of four different tetrapyridyl star connectors and 6 equiv of a platinum linker dication in nitromethane is presented. This face-directed approach affords high yields without template assistance. The prisms have been characterized by multinuclear and DOSY NMR and dual ESI-FT-ICR mass spectrometry. The use of a conformationally chiral star connector leads to a conformationally chiral prism when connector arm ends attached to a vertex have a strongly correlated twist sense and chirality is communicated across polyhedral faces, edges, and vertices. Molecular mechanics results suggest that in the smallest prism 3d collective effects dominate and the all-P and all-M conformers are strongly favored. NMR data prove that the two edges of the pyridine rings in the triflate salts of 3a-3d are distinct. An Eyring plot of rates obtained from line-shape analysis and 1-D EXCHSY NMR yields an activation enthalpy DeltaH(double dagger) of approximately 12 kcal/mol and activation entropy DeltaS(double dagger) of approximately -15 cal/mol x K for the edge interconversion process, compatible with pyridine rotation around the Pt-N bond. For 3c, this behavior is observed only up to approximately 318 K. At higher temperatures, the Eyring plot is again linear but follows a very different straight line, with a DeltaH(double dagger) of approximately 35 kcal/mol and DeltaS(double dagger) of approximately 60 cal/mol x K. This highly unusual result is further investigated and discussed in the following companion paper.

Development and characterization of an ionization technique for analysis of biological macromolecules: liquid matrix-assisted laser desorption electrospray ionization.

We have developed an atmospheric pressure ionization technique called liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI) for the generation of multiply charged ions by laser desorption from liquid samples deposited onto a stainless steel sample target biased at a high potential. This variant of our previously reported MALDESI source does not utilize an ESI emitter to postionize neutrals. Conversely, we report desorption and ionization from a macroscopic charged droplet. We demonstrate high mass resolving power single-acquisition FT-ICR-MS analysis of peptides and proteins ranging from 1 to 8.6 kDa at atmospheric pressure. The liquid sample acts as a macroscopic charged droplet similar to those generated by electrospray ionization, whereby laser irradiation desorbs analyte from organic matrix containing charged droplets generating multiply charged ions. We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization. Moreover, we demonstrate an increase in ion abundance and a concurrent decrease in surface tension with an increase in the applied potential.

Ambient aerodynamic ionization source for remote analyte sampling and mass spectrometric analysis.

The use of aerodynamic devices in ambient ionization source development has become increasingly prevalent in the field of mass spectrometry. In this study, an air ejector has been constructed from inexpensive, commercially available components to incorporate an electrospray ionization emitter within the exhaust jet of the device. This novel aerodynamic device, herein termed remote analyte sampling, transport, and ionization relay (RASTIR) was used to remotely sample neutral species in the ambient and entrain them into an electrospray plume where they were subsequently ionized and detected using a linear ion trap Fourier transform mass spectrometer. Two sets of experiments were performed in the ambient environment to demonstrate the device's utility. The first involved the remote (approximately 1 ft) vacuum collection of pure sample particulates (i.e., dry powder) from a glass slide, entrainment and ionization at the ESI emitter, and mass spectrometric detection. The second experiment involved the capture (vacuum collection) of matrix-assisted laser desorbed proteins followed by entrainment in the ESI emitter plume, multiple charging, and mass spectrometric detection. This approach is in principle a RASTIR-assisted matrix-assisted laser desorption electrospray ionization source (Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712-1716; Rapid Commun. Mass Spectrom. 2007, 21, 1150-1154.). A detailed description of the device construction, operational parameters, and preliminary small molecule and protein data are presented.

Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer.

Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.

Construction of a versatile high precision ambient ionization source for direct analysis and imaging.

The design and construction of a high precision ambient ionization source matrix-assisted laser desorption electrospray ionization (MALDESI) are described in full detail, including a complete parts list. The computer controlled high precision motion control system and high repetition rate Explorer laser are demonstrated during MALDESI-FT-ICR analysis of peptides and proteins ranging from 1 to 17 kDa. The high stability ionization source platform described herein demonstrates both the advantages of the new MALDESI source and versatility for application to numerous desorption and ionization techniques.