This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI) MALDI-MS was performed in the positive and negative ion modes using 2, 5-dihydroxy benzoic acid (20mg/1mL in 50% methanol) and 3, 5-dimethoxy-4-hydroxycinnamic acid (Sinapinic acid, saturated in H2O/ACN (1/1)) as a matrix. The sample (EPO Asn83) was diluted 2 fold in 0.1% TFA. The crystal was spotted with 0.5 L of sample and 0.5 L of matrix. Full mass spectra of sample were obtained initially using a MALDI TOF Mass Spectrometer (Applied Biosystems). NanoelectroSpray Ionization Linear Ion Trap mass Spectrometry (ESI-LTQ/MSn) Mass analysis by LTQ-MS (Thermo Finnigan) was performed by direct infusion of glycopeptide dissolved in 25 L of 0.1% formic acid and 25 L of 0.1% formic acid/80% acetonitrile into the LTQ instrument using a nanoelectrostray source at syringe flow rate of 0.40 L/min. The capillary temperature was set to 210oC and MS analysis was performed in positive ion mode. The collision energy for fragmentation in MS/MS was individually optimized for each MS signal.
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