Receptor deorphanization in an echinoderm reveals kisspeptin evolution and relationship with SALMFamide neuropeptides.

BACKGROUND: Kisspeptins are neuropeptides that regulate reproductive maturation in mammals via G-protein-coupled receptor-mediated stimulation of gonadotropin-releasing hormone secretion from the hypothalamus. Phylogenetic analysis of kisspeptin-type receptors indicates that this neuropeptide signaling system originated in a common ancestor of the Bilateria, but little is known about kisspeptin signaling in invertebrates. RESULTS: Contrasting with the occurrence of a single kisspeptin receptor in mammalian species, here, we report the discovery of an expanded family of eleven kisspeptin-type receptors in a deuterostome invertebrate - the starfish Asterias rubens (phylum Echinodermata). Furthermore, neuropeptides derived from four precursor proteins were identified as ligands for six of these receptors. One or more kisspeptin-like neuropeptides derived from two precursor proteins (ArKPP1, ArKPP2) act as ligands for four A. rubens kisspeptin-type receptors (ArKPR1,3,8,9). Furthermore, a family of neuropeptides that act as muscle relaxants in echinoderms (SALMFamides) are ligands for two A. rubens kisspeptin-type receptors (ArKPR6,7). The SALMFamide neuropeptide S1 (or ArS1.4) and a 'cocktail' of the seven neuropeptides derived from the S1 precursor protein (ArS1.1-ArS1.7) act as ligands for ArKPR7. The SALMFamide neuropeptide S2 (or ArS2.3) and a 'cocktail' of the eight neuropeptides derived from the S2 precursor protein (ArS2.1-ArS2.8) act as ligands for ArKPR6. CONCLUSIONS: Our findings reveal a remarkable diversity of neuropeptides that act as ligands for kisspeptin-type receptors in starfish and provide important new insights into the evolution of kisspeptin signaling. Furthermore, the discovery of the hitherto unknown relationship of kisspeptins with SALMFamides, neuropeptides that were discovered in starfish prior to the identification of kisspeptins in mammals, presents a radical change in perspective for research on kisspeptin signaling.

Tailor-made recombinant prokaryotic lectins for characterisation of glycoproteins.

Development of biosimilars is costly, where glycan analysis is a significant constraint on time and money. This paper provides an in-depth characterisation of several novel recombinant prokaryotic lectins (RPLs), developed through directed evolution, displaying specific binding activities to α-mannose, ß-galactose, fucose and sialic acid residues, tested against major biosimilar targets. The binding characterisation of all lectins was performed employing the principles of bio-layer interferometry (BLI), with help of the streptavidin-coated sensor with the biotinylated lectins. The binding activity of the RPLs and the specificity to a broad range of glycoproteins and glycoconjugates were evaluated and compared to those of equivalent plant-derived lectins. While exhibiting better or similar specificity, RPLs displayed significantly better binding in all cases. The binding mechanisms are explained with particular focus on the role hydrogen bonding plays in the change of specificity for a galactose specific lectin. Furthermore, different sets of RPLs and their plant equivalents were assayed against the different glycoprotein targets to evaluate the analytical parameters of the lectin-glycoprotein interaction. The obtained LoDs reached by the RPLs were lower than those of their plant counterparts apart from one, exhibiting RPL:PL LoD ratios of 0.8, 2.5, 14.2 and 380 for the sets of lectins specific to fucose, α-mannose, ß-galactose and sialic acid, respectively. Such enhancement in analytical parameters of RPLs shows their applicability in protein purification and as bioanalytical tools for glycan analysis and biosensor development.

Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm.

BACKGROUND: Vasopressin/oxytocin (VP/OT)-type neuropeptides are well known for their roles as regulators of diuresis, reproductive physiology and social behaviour. However, our knowledge of their functions is largely based on findings from studies on vertebrates and selected protostomian invertebrates. Little is known about the roles of VP/OT-type neuropeptides in deuterostomian invertebrates, which are more closely related to vertebrates than protostomes. RESULTS: Here, we have identified and functionally characterised a VP/OT-type signalling system comprising the neuropeptide asterotocin and its cognate G-protein coupled receptor in the starfish (sea star) Asterias rubens, a deuterostomian invertebrate belonging to the phylum Echinodermata. Analysis of the distribution of asterotocin and the asterotocin receptor in A. rubens using mRNA in situ hybridisation and immunohistochemistry revealed expression in the central nervous system (radial nerve cords and circumoral nerve ring), the digestive system (including the cardiac stomach) and the body wall and associated appendages. Informed by the anatomy of asterotocin signalling, in vitro pharmacological experiments revealed that asterotocin acts as a muscle relaxant in starfish, contrasting with the myotropic actions of VP/OT-type neuropeptides in vertebrates. Furthermore, in vivo injection of asterotocin had a striking effect on starfish behaviour-triggering fictive feeding where eversion of the cardiac stomach and changes in body posture resemble the unusual extra-oral feeding behaviour of starfish. CONCLUSIONS: We provide a comprehensive characterisation of VP/OT-type signalling in an echinoderm, including a detailed anatomical analysis of the expression of both the VP/OT-type neuropeptide asterotocin and its cognate receptor. Our discovery that asterotocin triggers fictive feeding in starfish provides important new evidence of an evolutionarily ancient role of VP/OT-type neuropeptides as regulators of feeding in animals.

Native electrospray mass spectrometry approaches to probe the interaction between zinc and an anti-angiogenic peptide from histidine-rich glycoprotein.

Zinc modulates the biological function of histidine-rich glycoprotein (HRG) through binding to its His-rich region (HRR). The Zn2+-binding properties of a 35 amino-acid biologically-active peptide mimic of the HRR, HRGP330, were investigated using dissociative mass spectrometry approaches in addition to travelling-wave ion mobility mass spectrometry (TWIM-MS). Native mass spectrometry confirmed zinc binding to HRGP330; however, broadening of the 1H NMR resonances upon addition of Zn2+ ions precluded the attainment of structural information. A complementary approach employing TWIM-MS indicated that HRGP330 has a more compact structure in the presence of Zn2+ ions. Top-down MS/MS data supported a metal-binding-induced conformational change, as fewer fragments were observed for Zn2+-bound HRGP330. Zn2+-bound fragments of both N-terminal and C-terminal ends of the peptide were identified from collision-induced dissociation (CID) and electron transfer dissociation/proton transfer reaction (ETD/PTR) experiments, suggesting that multiple binding sites exist within this region of HRG. The combination of mass spectrometry and NMR approaches provides new insight into the highly dynamic interaction between zinc and this His-rich peptide.

Discovery and functional characterisation of a luqin-type neuropeptide signalling system in a deuterostome.

Neuropeptides are diverse and evolutionarily ancient regulators of physiological/behavioural processes in animals. Here we have investigated the evolution and comparative physiology of luqin-type neuropeptide signalling, which has been characterised previously in protostomian invertebrates. Phylogenetic analysis indicates that luqin-type receptors and tachykinin-type receptors are paralogous and probably originated in a common ancestor of the Bilateria. In the deuterostomian lineage, luqin-type signalling has been lost in chordates but interestingly it has been retained in ambulacrarians. Therefore, here we characterised luqin-type signalling for the first time in an ambulacrarian - the starfish Asterias rubens (phylum Echinodermata). A luqin-like neuropeptide with a C-terminal RWamide motif (ArLQ; EEKTRFPKFMRW-NH2) was identified as the ligand for two luqin-type receptors in A. rubens, ArLQR1 and ArLQR2. Furthermore, analysis of the expression of the ArLQ precursor using mRNA in situ hybridisation revealed expression in the nervous system, digestive system and locomotory organs (tube feet) and in vitro pharmacology revealed that ArLQ causes dose-dependent relaxation of tube feet. Accordingly, previous studies have revealed that luqin-type signalling regulates feeding and locomotor activity in protostomes. In conclusion, our phylogenetic analysis combined with characterisation of luqin-type signalling in a deuterostome has provided new insights into neuropeptide evolution and function in the animal kingdom.

Travelling-wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N-glycans.

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1-acid glycoprotein and thyroglobulin were measured with a travelling-wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man3 GlcNAc3 from chicken ovalbumin and Man3 GlcNAc3 Fuc1 from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision-induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high-mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high-mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility-m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.

Urbilaterian origin of paralogous GnRH and corazonin neuropeptide signalling pathways.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive maturation in humans and other vertebrates. Homologs of GnRH and its cognate receptor have been identified in invertebrates-for example, the adipokinetic hormone (AKH) and corazonin (CRZ) neuropeptide pathways in arthropods. However, the precise evolutionary relationships and origins of these signalling systems remain unknown. Here we have addressed this issue with the first identification of both GnRH-type and CRZ-type signalling systems in a deuterostome-the echinoderm (starfish) Asterias rubens. We have identified a GnRH-like neuropeptide (pQIHYKNPGWGPG-NH2) that specifically activates an A. rubens GnRH-type receptor and a novel neuropeptide (HNTFTMGGQNRWKAG-NH2) that specifically activates an A. rubens CRZ-type receptor. With the discovery of these ligand-receptor pairs, we demonstrate that the vertebrate/deuterostomian GnRH-type and the protostomian AKH systems are orthologous and the origin of a paralogous CRZ-type signalling system can be traced to the common ancestor of the Bilateria (Urbilateria).

Global and Local Conformation of Human IgG Antibody Variants Rationalizes Loss of Thermodynamic Stability.

Immunoglobulin G (IgG) monoclonal antibodies (mAbs) are a major class of medicines, with high specificity and affinity towards targets spanning many disease areas. The antibody Fc (fragment crystallizable) region is a vital component of existing antibody therapeutics, as well as many next generation biologic medicines. Thermodynamic stability is a critical property for the development of stable and effective therapeutic proteins. Herein, a combination of ion-mobility mass spectrometry (IM-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) approaches have been used to inform on the global and local conformation and dynamics of engineered IgG Fc variants with reduced thermodynamic stability. The changes in conformation and dynamics have been correlated with their thermodynamic stability to better understand the destabilising effect of functional IgG Fc mutations and to inform engineering of future therapeutic proteins.

Protomers of benzocaine: solvent and permittivity dependence.

The immediate environment of a molecule can have a profound influence on its properties. Benzocaine, the ethyl ester of para-aminobenzoic acid that finds an application as a local anesthetic, is found to adopt in its protonated form at least two populations of distinct structures in the gas phase, and their relative intensities strongly depend on the properties of the solvent used in the electrospray ionization process. Here, we combine IR-vibrational spectroscopy with ion mobility-mass spectrometry to yield gas-phase IR spectra of simultaneously m/z and drift-time-resolved species of benzocaine. The results allow for an unambiguous identification of two protomeric species: the N- and O-protonated forms. Density functional theory calculations link these structures to the most stable solution and gas-phase structures, respectively, with the electric properties of the surrounding medium being the main determinant for the preferred protonation site. The fact that the N-protonated form of benzocaine can be found in the gas phase is owed to kinetic trapping of the solution-phase structure during transfer into the experimental setup. These observations confirm earlier studies on similar molecules where N- and O-protonation have been suggested.

Estimating collision cross sections of negatively charged N-glycans using traveling wave ion mobility-mass spectrometry.

Glycosylation is one of the most common post-translational modifications occurring in proteins. A detailed structural characterization of the involved carbohydrates, however, is still one of the greatest challenges in modern glycoproteomics, since multiple regio- and stereoisomers with an identical monosaccharide composition may exist. Recently, ion mobility-mass spectrometry (IM-MS), a technique in which ions are separated according to their mass, charge, and shape, has evolved as a promising technique for the separation and structural analysis of complex carbohydrates. This growing interest is based on the fact that the measured drift times can be converted into collision cross sections (CCSs), which can be compared, implemented into databases, and used as additional search criteria for structural identification. However, most of the currently used commercial IM-MS instruments utilize a nonuniform traveling wave field to propel the ions through the IM cell. As a result, CCS measurements cannot be performed directly and require calibration. Here, we present a calibration data set consisting of over 500 reference CCSs for negatively charged N-glycans and their fragments. Moreover, we show that dextran, already widely used as a calibrant in high performance liquid chromatography, is also a suitable calibrant for CCS estimations. Our data also indicate that a considerably increased error has to be taken into account when reference CCSs acquired in a different drift gas are used for calibration.

Fragmentation of negative ions from N-linked carbohydrates: part 6. Glycans containing one N-acetylglucosamine in the core.

RATIONALE: Negative ion collision-induced dissociation (CID) spectra of N-glycans contain many diagnostic ions that provide more structural information than positive ion spectra. EndoH or endoS release of glycans from glycoproteins, as used by many investigators, cleaves glycans between the GlcNAc residues of the chitobiose core leaving the glycan without the reducing-terminal GlcNAc residue. However, their negative ion CID spectra do not appear to have been studied in detail. This paper examines the CID and ion mobility properties of these endoH-released glycans to determine if the missing GlcNAc influences the production of diagnostic fragment ions. METHODS: N-Glycans were released from ribonuclease B, ovalbumin and gp120 with endoH to give high-mannose and hybrid glycans, and from IgG with endoS to produce biantennary complex glycans, all missing the reducing-terminal GlcNAc residue. Negative ion CID and travelling wave ion mobility spectra were recorded with a Waters Synapt G2 mass spectrometer using nanospray sample introduction. RESULTS: The majority of glycans yielded CID spectra exhibiting the same diagnostic fragments, which were equivalently informative, as the fully released structures. However, the ability of ion mobility to separate isomers was generally found to be inferior to its use with the full glycans despite the smaller nature of the compounds. The exception was the partial resolution of a pair of biantennary monogalactosylated glycans from IgG where, as chloride adducts, slight separation of the isomers was observed. CONCLUSIONS: The results show that the CID spectra of endoH- and endoS-released glycans are as useful as the corresponding spectra of the intact glycans (as released by PNGase F) in providing structural information on N-glycans.

Uukuniemi Phlebovirus assembly and secretion leave a functional imprint on the virion glycome.

Uukuniemi virus (UUKV) is a model system for investigating the genus Phlebovirus of the Bunyaviridae. We report the UUKV glycome, revealing differential processing of the Gn and Gc virion glycoproteins. Both glycoproteins display poly-N-acetyllactosamines, consistent with virion assembly in the medial Golgi apparatus, whereas oligomannose-type glycans required for DC-SIGN-dependent cellular attachment are predominant on Gc. Local virion structure and the route of viral egress from the cell leave a functional imprint on the phleboviral glycome.

Mass spectrometric strategies to improve the identification of Pt(II)-modification sites on peptides and proteins.

To further explore the binding chemistry of cisplatin (cis-Pt(NH3)2Cl2) to peptides and also establish mass spectrometry (MS) strategies to quickly assign the platinum-binding sites, a series of peptides with potential cisplatin binding sites (Met(S), His(N), Cys(S), disulfide, carboxyl groups of Asp and Glu, and amine groups of Arg and Lys, were reacted with cisplatin, then analyzed by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Radical-mediated side-chain losses from the charge-reduced Pt-binding species (such as CH3S(•) or CH3SH from Met, SH(•) from Cys, CO2 from Glu or Asp, and NH2(•) from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. The method was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin + Pt(NH3)2 + insulin (>10 kDa). In addition, ion mobility MS shows that Pt binds to multiple sites in Substance P, generating multiple conformers, which can be partially localized by collisionally activated dissociation (CAD). Platinum(II) (Pt(II)) was found to coordinate to amine groups of Arg and Lys, but not to disulfide bonds under the conditions used. The coordination of Pt to Arg or Lys appears to arise from the migration of Pt(II) from Met(S) as shown by monitoring the reaction products at different pH values by ECD. No direct binding of cisplatin to amine groups was observed at pH 3 ~ 10 unless Met residues were present in the sequence, but noncovalent interactions between cisplatin hydrolysis and amination [Pt(NH3)4](2+) products and these peptides were found regardless of pH.

Structural plasticity of the Semliki Forest virus glycome upon interspecies transmission.

Cross-species viral transmission subjects parent and progeny alphaviruses to differential post-translational processing of viral envelope glycoproteins. Alphavirus biogenesis has been extensively studied, and the Semliki Forest virus E1 and E2 glycoproteins have been shown to exhibit differing degrees of processing of N-linked glycans. However the composition of these glycans, including that arising from different host cells, has not been determined. Here we determined the chemical composition of the glycans from the prototypic alphavirus, Semliki Forest virus, propagated in both arthropod and rodent cell lines, by using ion-mobility mass spectrometry and collision-induced dissociation analysis. We observe that both the membrane-proximal E1 fusion glycoprotein and the protruding E2 attachment glycoprotein display heterogeneous glycosylation that contains N-linked glycans exhibiting both limited and extensive processing. However, E1 contained predominantly highly processed glycans dependent on the host cell, with rodent and mosquito-derived E1 exhibiting complex-type and paucimannose-type glycosylation, respectively. In contrast, the protruding E2 attachment glycoprotein primarily contained conserved under-processed oligomannose-type structures when produced in both rodent and mosquito cell lines. It is likely that glycan processing of E2 is structurally restricted by steric-hindrance imposed by local viral protein structure. This contrasts E1, which presents glycans characteristic of the host cell and is accessible to enzymes. We integrated our findings with previous cryo-electron microscopy and crystallographic analyses to produce a detailed model of the glycosylated mature virion surface. Taken together, these data reveal the degree to which virally encoded protein structure and cellular processing enzymes shape the virion glycome during interspecies transmission of Semliki Forest virus.

Discovery of a novel neurophysin-associated neuropeptide that triggers cardiac stomach contraction and retraction in starfish.

Feeding in starfish is a remarkable process in which the cardiac stomach is everted over prey and then retracted when prey tissue has been resorbed. Previous studies have revealed that SALMFamide-type neuropeptides trigger cardiac stomach relaxation and eversion in the starfish Asterias rubens. We hypothesized, therefore, that a counteracting neuropeptide system controls cardiac stomach contraction and retraction. Members of the NG peptide family cause muscle contraction in other echinoderms (e.g. NGFFFamide in sea urchins and NGIWYamide in sea cucumbers), so we investigated NG peptides as candidate regulators of cardiac stomach retraction in starfish. Generation and analysis of neural transcriptome sequence data from A. rubens revealed a precursor protein comprising two copies of a novel NG peptide, NGFFYamide, which was confirmed by mass spectrometry. A noteworthy feature of the NGFFYamide precursor is a C-terminal neurophysin domain, indicative of a common ancestry with vasopressin/oxytocin-type neuropeptide precursors. Interestingly, in precursors of other NG peptides the neurophysin domain has been retained (e.g. NGFFFamide) or lost (e.g. NGIWYamide and human neuropeptide S) and its functional significance remains to be determined. Investigation of the pharmacological actions of NGFFYamide in starfish revealed that it is a potent stimulator of cardiac stomach contraction in vitro and that it triggers cardiac stomach retraction in vivo. Thus, discovery of NGFFYamide provides a novel insight into neural regulation of cardiac stomach retraction as well as a rationale for chemically based strategies to control starfish that feed on economically important shellfish (e.g. mussels) or protected marine fauna (e.g. coral).

Travelling wave ion mobility and negative ion fragmentation for the structural determination of N-linked glycans.

Travelling wave ion mobility was investigated for its ability to separate N-glycans from other compounds and for resolution of isomers. Charged glycans, exemplified by sialylated complex N-glycans released from bovine fetuin and ionised by electrospray, could be separated from residual glycopeptides allowing the minor, more highly sialylated compounds to be detected where their ions were obscured by ions from other compounds in different charge states. This technique was also found to be excellent for extracting the N-glycan profiles from contaminated samples. Structural identification of the glycans was performed by negative ion CID fragmentation, a method that provides a wealth of structurally diagnostic ions. However, fragment ions can also appear in the glycan profiles where they can be mistaken for glycan molecular ions. Fragments and molecular ions were frequently shown to have different drift time profiles, allowing them to be differentiated. Some separation of isomers was found but only for the smallest compounds. Differentiation from conformers was achieved by plotting drift time profiles of the fragments; these profiles matched those of the precursor ions where conformers were present. The techniques were applied to investigations of N-glycans released from the fungus Piptoporus betulinus where the technique was used to separate different carbohydrate types present in biological extracts.

Is the higher risk of cardiovascular disease amongst South Asian populations linked to abnormalities of haemoglobin? A preliminary case control study.

The elevated burden of cardiovascular disease (CVD) amongst South Asian populations is a complex and multi-factorial phenomenon. South Asians evolved from environments where malaria was endemic, and while haemoglobin disorders frequent this group, a link to CVD has not been described. Using a case-control feasibility study, haemoglobin abnormalities identified by mass spectrometry were compared between South Asian patients with CVD (n = 72) and non-CVD controls (n = 84). Carotid-artery intima media thickness (CIMT) was used as a marker of vascular damage. Ultracentrifugation was used to separate lipoprotein subfractions, which were analysed for iron. Haemoglobin anomalies were more frequent for CVD patients than controls (34.7% vs. 14.3%, P < 0.001), as were subfractionated lipoprotein concentrations of iron (P < 0.001). Patients with haemoglobin disorders had greater CIMT (0.75 vs. 0.65 mm, P = 0.008), and lower HDL cholesterol (0.78 vs. 1.03 mmol/l, P = 0.003). These preliminary data suggest that haemoglobin disorders contribute to atherosclerotic disease in South Asians and further research is warranted.

Resolution of a paradox by native mass spectrometry: facile occupation of all four metal binding sites in the dimeric zinc sensor SmtB.

The predominant species of the cyanobacterial metalloregulatory protein SmtB as observed by ESI-MS is a dimer with all four zinc binding sites occupied.

MALDI-MS/MS with traveling wave ion mobility for the structural analysis of N-linked glycans.

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.

Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of Methylocella silvestris.

The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase-reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.