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Biomedical subjects

Y Sun

Publications and source records attributed to Y Sun.

At least 235 records · Page 13Linked to original sources

Ectopic scute induces Drosophila ommatidia development without R8 founder photoreceptors.

During development of the Drosophila peripheral nervous system, different proneural genes encoding basic helix-loop-helix transcription factors are required for different sensory organs to form. atonal (ato) is the proneural gene required for chordotonal organs and R8 photoreceptors, whereas the achaete-scute complex contains proneural genes for external sensory organs such as the macrochaetae, large sensory bristles. Whereas ectopic ato expression induces chordotonal organ formation, ectopic scute expression produces external sensory organs but not chordotonal organs in the wing. Proneural genes thus appear to specify the sensory organ type. In the ommatidium, or unit eye, R8 is the first photoreceptor to form and appears to recruit other photoreceptors and support cells. In the atonal(1) (ato(1)) mutant, R8 photoreceptors fail to form, thereby resulting in the complete absence of ommatidia. To our surprise, we found that ectopic scute expression in the ato(1) mutant induces the formation of ommatidia, which occasionally sprout ectopic macrochaetae. Remarkably, many scute-induced ommatidia lack R8 although they contain outer photoreceptors.

Animals↗

Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation.

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins. Oncogene (2000) 19, 2855 - 2866

Carrier Proteins↗

Lysozyme refolding with immobilized GroEL column chromatography.

A refolding chromatography with immobilized molecular chaperonin GroEL was studied for the reactivation of denatured-reduced lysozyme. The effect of denaturant concentration (guanidine hydrochloride, 0.1-1.5 M) in the elution buffer, the elution flow-rate, and the loading concentration and volume of the substrate protein on the reactivation yield was studied. All the operating parameters showed minor effects on the recovery yield of lysozyme mass, which remained at 90-100%, but exhibited relatively notable influences on the specific activity of the recovered lysozyme. For example, there existed an optimum denaturant concentration of about 1 M at which the highest yield of specific activity (up to 97%) was obtained. Using the immobilized GroEL column, 3 ml of the lysozyme (1 mg/ml) per batch could be refolded at an overall yield of 81%, which corresponded to a refolding productivity of 54 mg per 1 gel per h. At comparable reactivation yields (over 80%), this value of productivity was over four-times larger as that of the size-exclusion refolding chromatography reported previously (12 mg per 1 gel per h), indicating the advantage of the present system for producing a high throughput in protein refolding operations.

Chaperonin 60↗

4-Thiazolidinones: novel inhibitors of the bacterial enzyme MurB.

4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB. Selected 4-thiazolidinones displayed activity against the enzyme in vitro. This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.

Bacteria↗

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the gland's milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.

Animals↗

Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded DNA virus.

Scaffolding proteins are required for high fidelity assembly of most high T number dsDNA viruses such as the large bacteriophages, and the herpesvirus family. They function by transiently binding and positioning the coat protein subunits during capsid assembly. In both bacteriophage P22 and the herpesviruses the extreme scaffold C terminus is highly charged, is predicted to be an amphipathic alpha-helix, and is sufficient to bind the coat protein, suggesting a common mode of action. NMR studies show that the coat protein-binding domain of P22 scaffolding protein exhibits a helix-loop-helix motif stabilized by a hydrophobic core. One face of the motif is characterized by a high density of positive charges that could interact with the coat protein through electrostatic interactions. Results from previous studies with a truncation fragment and the observed salt sensitivity of the assembly process are explained by the NMR structure.

Amino Acid Sequence↗

Wild type and mutant p53 differentially regulate the gene expression of human collagenase-3 (hMMP-13).

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions including arthritis and cancer metastasis. Recently we have shown for the first time that the human MMP-1 gene is a p53 target gene subject to repression by wild type p53 (Sun, Y., Sun, Y. I., Wenger, L., Rutter, J. L., Brinckerhoff, C. E., and Cheung, H. S. (1999) J. Biol. Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down-regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter- and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis.

Arthritis, Rheumatoid↗

Generation of mammalian cells stably expressing multiple genes at predetermined levels.

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.

Animals↗

Synthesis and characterization of racemic mixture and meso isomers of bis(trans-2-aminocyclohexyl)aminepentaacetic acid and the stabilities of their Gd(III) complexes.

The synthesis and characterization of two multidentate ligands, the racemic mixture and meso isomers of bis-(trans-2-aminocyclohexyl)aminepentaacetic acid, are described. Equilibrium constants for their Gd(III) complexes were determined by direct potentiometry. The formation constants (KML = [ML]/[M][L]) of Gd(III)-Cycyracemic in 0.10 M KCl at 25.0 degrees C is 10(20.71; that for the meso isomer is 10(20.42). The crystal structure of (1S,2S,1'S,2'S)-bis(trans-2-aminocyclohexyl)amine-N,N,N',N",N"-pentaacetic acid, penta-tert-butyl ester (C42H75N3O10), is reported. This compound crystallizes in the triclinic system with space group P1 with cell parameters a = 10.805(2) A, b = 11.382(2) A, c = 20.999(4) A, alpha = 91.41(3) degrees, beta = 98.23(3) degrees, gamma = 113.88(3) degrees, V = 2327.8(8) A3, Z = 2, Dx = 1.116 g mL-1. The results are compared to the crystal structures of the gadolinium complexes and the predictions derived from molecular mechanics.

Algorithms↗

Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.

We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

Cloning, Molecular↗

Functional HLA-DM on the surface of B cells and immature dendritic cells.

HLA-DM (DM) plays a critical role in antigen presentation through major histocompatibility complex (MHC) class II molecules. DM functions as a molecular chaperone by keeping class II molecules competent for antigenic peptide loading and serves as an editor by favoring presentation of high-stability peptides. Until now, DM has been thought to exert these activities only in late endosomal/lysosomal compartments of antigen-presenting cells. Here we show that a subset of DM resides at the cell surface of B cells and immature dendritic cells. Surface DM engages in complexes with putatively empty class II molecules and controls presentation of those antigens that rely on loading on the cell surface or in early endosomal recycling compartments. For example, epitopes derived from myelin basic protein that are implicated in the autoimmune disease multiple sclerosis are down-modulated by DM, but are presented in the absence of DM. Thus, this novel concept of functional DM on the surface may be relevant to both protective immune responses and autoimmunity.

Amino Acid Sequence↗

Helicobacter pylori-antigen-binding fragments expressed on the filamentous M13 phage prevent bacterial growth.

Colonization of the human stomach by Helicobacter pylori is associated with the development of gastritis, duodenal ulcer, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori-antigen-binding single-chain variable fragments (ScFv) were derived from murine hybridomas producing monoclonal antibodies and expressed as a g3p-fusion protein on a filamentous M13 phage. The recombinant ScFv-phage reacted specifically with a 30-kDa monomeric protein of a H. pylori surface antigen preparation and by means of immunofluorescence microscopy the phage was shown to bind to both the spiral and coccoid forms of the bacterium. In vitro, the recombinant phage exhibited a bacteriocidal effect and inhibited specifically the growth of all the six strains of H. pylori tested. When H. pylori was pretreated with the phage 10 min before oral inoculation of mice, the colonization of the mouse stomachs by the bacterium was significantly reduced (P<0.01). The results suggest that genetic engineering may be used to generate therapy-effective phages.

Animals↗

Comparative effects of pretreatment with captopril and losartan on cardiovascular protection in a rat model of ischemia-reperfusion.

OBJECTIVES: We sought to assess the comparative effects of pretreatment with captopril and losartan on myocardial infarct size and arrhythmias in a rat model of ischemia-reperfusion. BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) inhibit the renin-angiotensin system in different ways. However, the comparative effects of pretreatment with ACE inhibitors or ARBs on acute myocardial infarct size and arrhythmias are unknown. METHODS: We randomly assigned 117 female Sprague-Dawley rats into three groups: group N was the normal control; group C was given 40 mg/kg body weight per day of captopril in drinking water; and group L was given 40 mg/kg per day of losartan in drinking water. After 10 weeks of pretreatment, 25 rats in each group were subjected to 17 min of left anterior descending coronary artery occlusion and 2 h of reperfusion with hemodynamic and electrocardiographic monitoring. Fourteen rats in each group had blood samples drawn and aortic rings removed to study vascular reactivity. RESULTS: Mortality during ischemia and reperfusion was lower in combined groups L and C than in group N (4.2% vs. 19.2%, p = 0.042). Rats treated with losartan had significantly higher levels of angiotensin II in their plasma. Hemodynamic variables were not significantly different among the three groups. The thresholds of ventricular fibrillation (VF) before occlusion and after reperfusion were significantly higher in groups L and C than in group N (1.99 +/- 0.24 and 1.93 +/- 0.27 vs. 1.23 + 0.17 mA, p = 0.04; 2.13 +/- 0.25 and 1.78 +/- 0.22 vs. 0.95 +/- 0.11 mA, p = 0.001). The average episodes of ventricular tachycardia (VT) and VF per rat were significantly less in groups L and C than in group N (0.96 +/- 0.2 and 1.2 +/- 0.3 vs. 2.8 + 0.4 mA, p < 0.001). Myocardial infarct size was significantly smaller in groups L and C than in group N (34 +/- 3% and 35 +/- 3% vs. 44 +/- 3%, p = 0.031, 0.043). Endothelium-dependent vasorelaxation induced by a calcium ionophore (A23187) was increased in both groups but was only statistically significant in group C (p = 0.020). CONCLUSIONS: Losartan and captopril have similar cardiovascular protective effects in a rat model of ischemia-reperfusion. They increased the threshold of VF, decreased mortality and decreased episodes of VT and VF, as well as decreased myocardial infarct size.

Angiotensin II↗

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism.

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Animals↗

beta-arrestin differentially regulates the chemokine receptor CXCR4-mediated signaling and receptor internalization, and this implicates multiple interaction sites between beta-arrestin and CXCR4.

The chemokine receptor CXCR4 has recently been shown to be a co-receptor involved in the entry of human immunodeficiency virus type 1 into target cells. This study shows that coexpression of beta-arrestin with CXCR4 in human embryonic kidney 293 cells attenuated chemokine-stimulated G protein activation and inhibition of cAMP production. Truncation of the C-terminal 34 amino acids of CXCR4 (CXCR4-T) abolished the effects of beta-arrestin on CXCR4/G protein signaling, indicating the functional interaction of the receptor C terminus with beta-arrestin. On the other hand, receptor internalization and the subsequent activation of extracellular signal-regulated kinases were significantly promoted by coexpression of beta-arrestin with CXCR4, whereas the C-terminal truncation of CXCR4 did not affect this regulation of beta-arrestin, suggesting that beta-arrestin can functionally interact with CXCR4 with or without the C terminus. Moreover, beta(2)V54D, the dominant inhibitory mutant of beta-arrestin 2, exerted no effects on CXCR4/G protein signaling, but strongly influenced receptor internalization and extracellular signal-regulated kinase activation. Further cross-linking experiments demonstrated that beta-arrestin as well as beta(2)V54D could physically contact both CXCR4 and CXCR4-T. Glutathione S-transferase pull-down assay showed that beta-arrestin was able to bind efficiently in vitro to both the third intracellular loop and the 34-amino acid C terminus of CXCR4. Taken together, our data clearly establish that beta-arrestin can effectively regulate different functions of CXCR4 and that this is mediated through its distinct interactions with the C terminus and other regions including the third loop of CXCR4.

Arrestins↗

PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member, is a p53 target gene that inhibits tumor cell growth via TGF-beta signaling pathway.

Identification and characterization of p53 target genes would lead to a better understanding of p53 functions and p53-mediated signaling pathways. Two putative p53 binding sites were identified in the promoter of a gene encoding PTGF-beta, a type beta transforming growth factor (TGF-beta) superfamily member. Gel shift assay showed that p53 bound to both sites. Luciferase-coupled transactivation assay revealed that the gene promoter was activated in a p53 dose- as well as p53 binding site-dependent manner by wild-type p53 but not by several p53 mutants. The p53 binding and transactivation of the PTGF-beta promoter was enhanced by etoposide, a p53 activator, and was largely blocked by a dominant negative p53 mutant. Furthermore, expression of endogenous PTGF-beta was remarkably induced by etoposide in p53-positive, but not in p53-negative, cell lines. Finally, the conditioned medium collected from PTGF-beta-overexpressing cells, but not from the control cells, suppressed tumor cell growth. Growth suppression was not, however, seen in cells that lack functional TGF-beta receptors or Smad4, suggesting that PTGF-beta acts through the TGF-beta signaling pathway. Thus, PTGF-beta, a secretory protein, is a p53 target that could mediate p53-induced growth suppression in autocrinal as well as paracrinal fashions. The finding made a vertical connection between p53 and TGF-beta signaling pathways in controlling cell growth and implied a potential important role of p53 in inflammation regulation via PTGF-beta.

Binding Sites↗

Gene transfer to liver cancer cells of B7-1 plus interleukin 12 changes immunoeffector mechanisms and suppresses helper T cell type 1 cytokine production induced by interleukin 12 alone.

To investigate the cooperative effect of B7-1 and IL-12 in the induction of antitumor activity, we have developed retroviral vectors encoding human B7-1, murine IL-12, or both B7-1 and IL-12 coordinately. Murine transformed liver cells (BNL) were engineered to stably express B7-1, IL-12, or both by infection with corresponding retroviruses. No tumor was observed in 20, 75, and 95% of mice receiving, respectively, B7-1-, IL-12-, and B7-1/IL-12-modified tumor cells after 250 days of inoculation. In contrast, injection of parental BNL or BNL/Neo cells resulted in lethal tumor progression in all mice. Protection against rechallenge with parental tumor cells was observed only in mice who had rejected BNL/IL-12, but not in animals that rejected BNL/B7-1 or BNL/B7-1-IL-12. Growth of parental tumor cells was significantly delayed by simultaneous injection in a distant site of irradiated tumor cells engineered to express IL-12 or both B7-1 and IL-12 but not B7-1 alone. BNL/B7-1 and BNL/B7-1-IL-12 showed similar efficacy in these experiments. Antitumor immunity induced by B7, with or without IL-12, was found to depend mainly on CD4+ T cells with a minor contribution of a non-T cell mechanism; whereas the effect of IL-12 was dependent on CD8+ T cells and on non-T cell effectors. Immunization of mice with IL-12-modified BNL cells induced secretion of a Thl pattern of cytokines while immunization with cells expressing both IL-12 and B7-1 resulted in inhibition of IFN-gamma production. Immunization with BNL/B7-1-IL-12 cells in the presence of anti-human B7-1 MAb resulted in restoration of IFN-gamma production to the levels found in animals injected with BNL/IL-12 cells. To summarize, in our model coexpression of B7-1 and IL-12 in tumor cells does not result in improved antitumoral activity as compared with expression of IL-12 alone. This may be related to the fact that B7-1 changes the mechanisms of antitumor immunity and inhibits IFN-gamma production induced by IL-12 in vivo.

Animals↗

Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.

Journal Article↗