4-sulfophenyl Isothiocyanate Research Articles

Single Cell Peptidomics: Approach for Peptide Identification by N-Terminal Peptide Derivatization.

In recent years, single cell microanalysis techniques have moved into the center stage to study fundamental intracellular interactions and cell-cell communication events, and have led to a better understanding of physiological processes and behavioral patterns. The availability of more sensitive, robust, and precise mass spectrometers improved the detection and characterization of putative neuroactive substances from individual cells. For sequence characterization, particularly when working with samples as small as a single cell, the most crucial step to obtain usable data is sample preparation. For some studies, genetic or molecular data are not available to confirm an amino acid sequence of a putative neuropeptide, and it is necessary to sequence the peptide from the mass spectrometry analysis alone (i.e., de novo sequencing). In this chapter, a protocol is described for de novo sequencing of neuropeptides from individual single cells by N-terminal derivatization using 4-sulfophenyl isothiocyanate and subsequent mass spectrometric analysis.

Modulation of Phosphopeptide Fragmentation via Dual Spray Ion/Ion Reactions Using a Sulfonate-Incorporating Reagent.

The labile nature of phosphoryl groups has presented a long-standing challenge for the characterization of protein phosphorylation via conventional mass spectrometry-based bottom-up proteomics methods. Collision-induced dissociation (CID) causes preferential cleavage of the phospho-ester bond of peptides, particularly under conditions of low proton mobility, and results in the suppression of sequence-informative fragmentation that often prohibits phosphosite determination. In the present study, the fragmentation patterns of phosphopeptides are improved through ion/ion-mediated peptide derivatization with 4-formyl-1,3-benezenedisulfonic acid (FBDSA) anions using a dual spray reactor. This approach exploits the strong electrostatic interactions between the sulfonate moieties of FBDSA and basic sites to facilitate gas-phase bioconjugation and to reduce charge sequestration and increase the yield of phosphate-retaining sequence ions upon CID. Moreover, comparative CID fragmentation analysis between unmodified phosphopeptides and those modified online with FBDSA or in solution via carbamylation and 4-sulfophenyl isothiocyanate (SPITC) provided evidence for sulfonate interference with charge-directed mechanisms that result in preferential phosphate elimination. Our results indicate the prominence of charge-directed neighboring group participation reactions involved in phosphate neutral loss, and the implementation of ion/ion reactions in a dual spray reactor setup provides a means to disrupt the interactions by competing hydrogen-bonding interactions between sulfonate groups and the side chains of basic residues.

High fidelity approach for proteomic scale enrichment and identification of N-termini

A novel workflow was designed for the large-scale identification of protein N-terminal sequences. The workflow started with converting lysine to homoarginine, followed by reaction with sulfonation of N-termini by 4-sulfophenyl isothiocyanate (SPITC). Upon trypsin digestion, the SPITC modified N-terminal peptides were enriched by electrostatic repulsion hydrophilic interaction (ERLIC) chromatography in which the internal and C-terminal peptides eluted at the void volume, and the SPITC peptides were retained in the column due to the hydrophilicity and electrostatic attraction of the sulfonyl group to the stationary phase. Upon higher-energy collisional induced dissociation (HCD) SPITC peptides in ESI generated predominately y-type ion series; such simplification of spectra enables the identification of N-termini with high confidence. The appearance of b1+SPITC product ions upon HCD further boosts the confidence for N-terminal identifications. This method was applied to an Escherichia coli cell lysate, thus allowing the identification of 358 high confidence N-terminal peptides representing 274 distinct E. coli proteins as certain proteins were found to have multiple N-terminal peptides due to cleavage from various cellular enzymes. Confidence in N-terminal assignments is further heightened since 224 of the unique proteins identified from N-terminal peptides were also identified in the analysis of flow-through fractions.

Preparation of Chemically-Tailored Copolymer Membranes with Tunable Ion Transport Properties.

Membranes derived from copolymer materials are a promising platform due to their straightforward fabrication and small yet tunable pore structures. However, most current applications of these membranes are limited to the size-selective filtration of solutes. In this study, to advance the utility of copolymer membranes beyond size-selective filtrations, a poly(acrylonitrile-r-oligo(ethylene glycol) methyl ether methacrylate-r-glycidyl methacrylate) (P(AN-r-OEGMA-r-GMA)) copolymer is used to fabricate membranes that can be chemically modified via straightforward schemes. The P(AN-r-OEGMA-r-GMA) copolymer is cast into asymmetric membranes using a nonsolvent induced phase separation technique. Then, the surface charge of the membrane is modified to tailor its performance for nanofiltration applications. The oxirane groups of the glycidyl methacrylate (GMA) moiety that line the pore walls of the membrane allows for both positively charged and negatively charged moieties to be introduced directly without any prior activation. Notably, the highly size-selective nanostructure of the copolymer materials is retained throughout the functionalization processes. Specifically, amine moieties are attached to the pore walls using the aminolysis of the oxirane groups. The resulting amine-functionalized membrane is positively charged and rejects up to 87% of the salt dissolved in a 10 mM magnesium chloride feed solution. Further modification of the amine-functionalized membrane with 4-sulfophenyl isothiocyanate results in pore walls lined by sulfonic acid moieties. These negatively charged membranes reject up to 90% of a 10 mM sodium sulfate feed solution. Throughout the modification scheme, the membrane permeability remains equal to 1.5 L m(-2) h(-1) bar(-1) and the rejection of neutral solutes (i.e., sucrose and poly(ethylene oxide)) is consistent with the membrane having a single well-defined pore diameter of ∼5 nm. The performance of the membrane as a function of ion valence number, solution pH, and ionic strength is investigated.

Manipulation of cellulose nanocrystal surface sulfate groups toward biomimetic nanostructures in aqueous media

We report a facile aqueous procedure to create multivalent displays of sulfonated ligands on CNCs for future applications as viral inhibitors. CNCs were decorated with model compounds containing sulfonate groups via reactions of epoxides and isothiocyanates with amines under alkaline conditions. At first, surface sulfate groups of CNCs were hydrolytically cleaved by alkaline hydrolysis to increase the number of available surface hydroxyls. Success of desulfation was confirmed via dynamic light scattering (DLS), zeta potential measurements and thermogravimetric analysis (TGA). CNC surface hydroxyl groups were then activated with epichlorohydrin before subsequent reactions. As proof of concept toward aqueous pathways for functionalizing nanoparticles with sulfonated ligands, 3-chloro-2-hydroxy-1-propanesulfonic acid sodium salt hydrate (CPSA) and 4-sulfophenyl isothiocyanate sodium salt monohydrate (4-SPITC) were chosen as model compounds to react with homobifunctional 2,2′-(ethylenedioxy)bis(ethylamine) (EBEA) molecular spacer. The approaches presented are not only applicable to polysaccharide nanocrystals, but also other classes of polymeric and inorganic substrates presenting surface hydroxyl groups, as in the case of poly(2-hydroxyethyl methacrylate) (PHEMA), silica or glass. CNCs carrying sulfonated ligands were characterized by ATR-FTIR and UV–vis spectroscopy. Surface chemical compositions of desired elements were determined via X-ray photoelectron spectroscopy (XPS). We anticipate that with these facile aqueous procedures as the proof of concept, a diverse library of target-specific functionalities can be conjugated to CNCs for applications in nanomedicine, especially related to viral inhibition.

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.

Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

BackgroundDespite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach.ResultsThe cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses.ConclusionsThe proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.

Simple chemical tools to expand the range of proteomics applications

Proteomics is an expanding technology with potential applications in many research fields. Even though many research groups do not have direct access to its main analytical technique, mass spectrometry, they can interact with proteomics core facilities to incorporate this technology into their projects. Protein identification is the analysis most frequently performed in core facilities and is, probably, the most robust procedure. Here we discuss a few chemical reactions that are easily implemented within the conventional protein identification workflow. Chemical modification of proteins with N-hydroxysuccinimide esters, 4-sulfophenyl isothiocyanate, O-methylisourea or through β-elimination/Michael addition can be easily performed in any laboratory. The reactions are quite specific with almost no side reactions. These chemical tools increase considerably the number of applications and have been applied to characterize protein-protein interactions, to determine the N-terminal residues of proteins, to identify proteins with non-sequenced genomes or to locate phosphorylated and O-glycosylated.

Enhancement of Ultraviolet Photodissociation Efficiencies through Attachment of Aromatic Chromophores

Two N-terminal derivatization reagents containing aromatic chromophores, 4-sulfophenyl isothiocyanate (SPITC) and 4-methylphosphonophenyl isothiocyanate (PPITC), were used to increase the dissociation efficiencies of peptides upon ultraviolet photodissociation (UVPD) at 193 nm. The resulting UVPD spectra are dominated by C-terminal ions, including y, z, x, v, and w ions, and immonium ions. The attachment of the PPITC or SPITC groups leads to a reduction in the number and abundances of N-terminal ions because the added phosphonate or sulfonate functionalities result in neutralization of some of the N-terminal species, ones that might normally be singly protonated in the absence of the negatively charged sulfonate or phosphonate groups. In addition, the greater photoabsorptivities of the PPITC- and SPITC-derivatized N-terminal product ions enhanced their secondary photodissociation, leading to formation of immonium ions.

Protein Quantification by MALDI-Selected Reaction Monitoring Mass Spectrometry Using Sulfonate Derivatized Peptides

The feasibility of protein absolute quantification with matrix-assisted laser desorption/ionization (MALDI) using the selected reaction monitoring (SRM) acquisition mode on a triple quadrupole linear ion trap mass spectrometer (QqQ(LIT)) equipped with a high-frequency laser is demonstrated. A therapeutic human monoclonal antibody (mAb) was used as a model protein, and four tryptic peptides generated by fast tryptic digestion were selected as quantification surrogates. MALDI produces mostly singly charged peptides which hardly fragment under low-energy collision-induced dissociation (CID), and therefore the benefits of using 4-sulfophenyl isothiocyanate (SPITC) as a fragmentation enhancer derivatization agent were evaluated. Despite a moderate impact on the sensitivity, the N-terminus sulfonated peptides generate nearly complete y-ion ladders when native peptides produce few fragments. This aspect provides an alternative SRM transition set for each peptide. As a consequence, SRM transitions selectivity can be tuned more easily for peptide quantitation in complex matrices when monitoring several SRM transitions. From a quantitative point of view, the signal response depending on mAb concentration was found to be linear over 2.5 orders of magnitude for the most sensitive peptide, allowing precise and accurate measurement by MALDI-SRM/MS.

Zif, the Zoocin A Immunity Factor, Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a D-alanyl-L-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three L-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.

N-Terminal Derivatization of Peptides with Isothiocyanate Analogues Promoting Edman-Type Cleavage and Enhancing Sensitivity in Electrospray Ionization Tandem Mass Spectrometry Analysis

N-terminal derivatization of peptides with Edman's reagent, phenyl isothiocyanate (PITC), promotes gas-phase Edman cleavage that yields abundant complementary b(1) and y(n-1) ion pairs by tandem mass spectrometry (MS/MS). The formation of b(1) ions can be utilized as a mass tag to enhance the interpretation of MS/MS spectra and increase the confidence of peptide identification during mass spectrometry analysis. Derivatization of tryptic peptides with another isothiocyanate analogue, 4-sulfophenyl isothiocyanate, also produces signature ions resulting from Edman cleavage and facilitates peptide sequencing on linear or branched peptides. The limitation of these derivatizations, however, is reduced MS signal intensities of modified peptides, due presumably to the tags themselves. Here we have demonstrated that several other isothiocyanate analogues bearing basic moieties can derivatize peptides and significantly improve the MS sensitivity of tagged analytes, while promoting Edman fragmentation and maintaining other sequence fragments as well.

De novo sequence analysis and intact mass measurements for characterization of phycocyanin subunit isoforms from the blue‐green alga Aphanizomenon flos‐aquae

In this work, partial characterization of the primary structure of phycocyanin from the cyanobacterium Aphanizomenon flos-aquae (AFA) was achieved by mass spectrometry de novo sequencing with the aid of chemical derivatization. Combining N-terminal sulfonation of tryptic peptides by 4-sulfophenyl isothiocyanate (SPITC) and MALDI-TOF/TOF analyses, facilitated the acquisition of sequence information for AFA phycocyanin subunits. In fact, SPITC-derivatized peptides underwent facile fragmentation, predominantly resulting in y-series ions in the MS/MS spectra and often exhibiting uninterrupted sequences of 20 or more amino acid residues. This strategy allowed us to carry out peptide fragment fingerprinting and de novo sequencing of several peptides belonging to both alpha- and beta-phycocyanin polypeptides, obtaining a sequence coverage of 67% and 75%, respectively. The presence of different isoforms of phycocyanin subunits was also revealed; subsequently Intact Mass Measurements (IMMs) by both MALDI- and ESI-MS supported the detection of these protein isoforms. Finally, we discuss the evolutionary importance of phycocyanin isoforms in cyanobacteria, suggesting the possible use of the phycocyanin operon for a correct taxonomic identity of this species.

Use of 4-sulfophenyl isothiocyanate labeling and mass spectrometry to determine the site of action of the streptococcolytic peptidoglycan hydrolase zoocin A.

Zoocin A is a streptococcolytic peptidoglycan hydrolase with an unknown site of action that is produced by Streptococcus equi subsp. zooepidemicus 4881. Zoocin A has now been determined to be a d-alanyl-l-alanine endopeptidase by digesting susceptible peptidoglycan with a combination of mutanolysin and zoocin A, separating the resulting muropeptides by reverse-phase high-pressure liquid chromatography, and analyzing them by mass spectrometry (MS) in both the positive- and negative-ion modes to determine their compositions. In order to distinguish among possible structures for these muropeptides, they were N-terminally labeled with 4-sulfophenyl isothiocyanate (SPITC) and analyzed by tandem MS in the negative-ion mode. This novel application of SPITC labeling and MS/MS analysis can be used to analyze the structure of peptidoglycans and to determine the sites of action of other peptidoglycan hydrolases.

On‐target sample preparation of 4‐sulfophenyl isothiocyanate‐derivatized peptides using AnchorChip Targets

De novo sequencing of tryptic peptides by post source decay (PSD) or collision induced dissociation (CID) analysis using MALDI TOF-TOF instruments is due to the easy interpretation facilitated by the introduction of N-terminal sulfonated derivatives. Recently, a stable and cheap reagent, 4-sulfophenyl isothiocyanate (SPITC), has been successfully used for N-terminal derivatization. Previously described methods have always used desalting and concentration by reverse-phase chromatography prior to mass spectrometric analysis. Here we present an on-target sample preparation method based on AnchorChip target technology. The method was optimized for reduction of by-products and sensitivity with SPITC-derivatized tryptic BSA peptides, and successfully applied to protein identification from silver-stained two-dimensional electrophoretic gels of fish liver extracts. The method is simple and sensitive and allowed protein identification based on de novo sequencing and BLAST search from species with limited sequence information.

Improved protein identification efficiency by mass spectrometry using N‐terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.

Improved protein identification efficiency by mass spectrometry using N‐terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.

Observation of an unusually facile fragmentation pathway of gas‐phase peptide ions: a study on the gas‐phase fragmentation mechanism and energetics of tryptic peptides modified with 4‐sulfophenyl isothiocyanate (SPITC) and 4‐chlorosulfophenyl isocyanate (SPC) and their 18‐crown‐6 complexes

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.

Sequencing of phosphopeptides sulfonated by 4-sulfophenyl isothiocyanate using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract A combination of carbon isotope composition and stomatal characters of fossil leaves provides information about paleoatmospheric CO2 levels and the physiology of fossil plants. Plant anatomy, physiology, and geochemistry have been employed to reconstruct the paleoatmospheric CO2 concentrations throughout Jurassic time and to investigate the physiological response of fossil Ginkgo to an atmosphere of much higher CO2 than usual. Our results show that fossil Ginkgo in China lived in an atmosphere with a CO2 concentration 3–5 times higher than that of today. It used a carbon isotopic discrimination similar to that of the living plant, but had much higher water use efficiency (3–5 times that of the extant). They physiological traits of Ginkgo in different atmospheres suggest that CO2 might have played a contributory role in the rise and fall of maidenhair trees. *Supported by National Natural Science Foundation of China (Grant No. 40372012, 40502005), the key project of chinese Ministry of Educa-tion ...

Detection of hypoxia-related proteins in medaka ( Oryzias latipes) brain tissue by difference gel electrophoresis and de novo sequencing of 4-sulfophenyl isothiocyanate-derivatized peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Two-dimensional fluorescence-based difference gel electrophoresis (DIGE) was used in combination with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI–TOF/TOF-MS) to identify a set of hypoxia-related biomarker proteins in medaka ( Oryzias latipes) brain tissue. Each of the proteins were identified via de novo sequencing of tryptic peptides derivatized with 4-sulfophenyl isothiocyanate (SPITC), which N-terminally sulfonates peptides and promotes facile post-source decay peptide fragmentation, resulting in greatly simplified spectra consisting mainly of y-series fragment ions. We also report that addition of the non-ionic surfactant n-octyl-β- d-glucopyranoside significantly improves SPITC-derivatized peptide recoveries. In addition, we found that a MALDI matrix consisting of the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone, diammonium citrate, and α-cyano-4-hydroxycinnamic acid also improves ionization of SPITC-peptides, presumably by reducing ionization suppression effects from matrix contaminants, especially sodium cations. The DIGE experiments and analyses resulted in detection of six abundant proteins and related isozymes up-regulated (> 1.49, p < 0.005) in hypoxic medaka brain tissues, including two hemoglobin β subunit forms, four carbonic anhydrase 2 forms, calbindin, aldolase, succinate dehydrogenase, and glutathione- S-transferase.