br After the removal of N glycan

After the removal of N-glycan, as previously described in “1. N-glycomic strategy I: Sample preparation and LC-ESI-MS/MS”, the O-linked glycans were released from retained glycoproteins in spinfilter using reductive β-elimination (0.5M NaBH4, 50mM NaOH at 50°C, 16h). Reactions were quenched with 1µl of glacial acetic pannexin-1 inhibitor and glycan samples were desalted and dried as previously described [3]. Glycans were subjected to LC-ESI-MS/MS analysis using a 10cmx250µm I.D. column, prepared in-house, containing 5µm porous graphitized carbon (PGC) particles (Thermo Scientific. Waltham. MA). Glycans were eluted using a linear gradient from 0% to 40% acetonitrile in 10mM NH4HCO3 over 40min at a flow rate of 10μl/min. The eluted O-glycans were detected using a LTQ ion trap mass spectrometer (Thermo Scientific) in negative-ion mode with an electrospray voltage of 3.5kV, capillary voltage of −33.0V and capillary temperature of 300°C. Air was used as a sheath gas and mass ranges were defined dependent on the specific structure to be analyzed. The data were processed using the Xcalibur software (version 2.0.7. Thermo Scientific) and manually interpreted from their MS/MS spectra (Table 3).
Sample preparation and analysis were performed as previously described in Section 2. “N-glycomic strategy II: Sample preparation and PGC nanoLC-ESI MS/MS” (Table 4).
Sialoproteomic analysis
Acknowledgments We acknowledge the support from the European Union, Seventh Framework Programme, Gastric Glyco Explorer Initial Training Network: Grant number 316929. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology. This work is funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (FCOMP-01-0124-FEDER028188) and National Funds through the FCT-Foundation for Science and Technology, under the projects: PEst-C/SAU/LA0003/2013, PTDC/BBB-EBI/0786/2012, PTDC/BBB-EBI/0567/2014 (CR). This work was also supported by \"Glycoproteomics\" project Grant number PCIG09-GA-2011-293847 (to DK) and the Danish Natural Science Research Council and a generous Grant from the VILLUM Foundation to the VILLUM Center for Bioanalytical Sciences at the University of Southern Denmark (to MRL). AM acknowledges FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu) (SFRH/BPD/75871/2011). The UPLC instrument was obtained with a grant from the Ingabritt and Arne Lundbergs Research Foundation. C.J. was supported by the Knut and Alice Wallenberg Foundation. The mass spectrometer (LTQ) was obtained by a grant from the Swedish Research Council (342-2004-4434).
Data The data in Tables 1–3 were acquired by analyzing the spectra related to the spots highlighted in the 2-D protein profiles of the saliva of the triatominae Rhodnius prolixus, Triatoma lecticularia and Panstrongylus herreri. Table 1 refers to all proteins found in each spot for the three species. It shows the following characteristics for each protein: species, spot related to the electrophoretic profile, UniProt code, protein name, protein group, isoelectric point (pI) and molecular weight (MW) for each electrophoretic profile, molecular mass referring to UniProt, volume percentage referring to the spot of the electrophoretic profile (vol%), number of peptides (#Pep) and percent coverage found by mass spectrometry related to that particular protein. Table 2 refers to the most significant protein per spot (MSP) for the three species under study. It shows the same characteristics shown in Table 1 for each protein. Table 3 refers to the proteins found in the studied species according to biological process and molecular function.
Experimental design, materials and methods
Acknowledgments
Value of the data
Data We have identified dominant, temperature-sensitive mutations within the Drosophila type IV collagen gene col4a1, the insect homologue of mammalian COL4A1. Similar to their mammalian counterparts, the mutations trigger a systemic phenotype, including severe myopathy, intestinal dysfunction and a robust immune response manifested by overexpression of antimicrobial peptides and excess synthesis of the oxidants hydrogen peroxide and peroxynitrite [1–3]. The Malpighian tubules, the excretory organ of insects, are functionally similar to the mammalian kidneys [4]. The tubules are freely floating within the hemocoel, the openly circulating blood-filled body cavity. We surmised that the mechanical impetus of periodic movements of the insect body that keeps the Malpighian tubules in continuous movement, may also contribute to stress-induced cytoskeletal reorganization in mutant animals.