Thermo Scientific Hypercarb Columns

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B IOCHEMICALApplication NotesFigure 3: Comparison of different charge state extracted ion chromatograms of bovine α1-acid glycoprotein bi-antennary peptide 91 CVYNCSFIK 99 using C18 andporous graphite columnsFigure 4: Deconvoluted full MS spectrum of bovine α1-acid glycoprotein bi-antennary peptide 91 CVYNCSFIK 9912
Type of LC ColumnPeptides Graphite C18 C8103 QNGTLSK 109 •53 NPEYNK 58 •91 CVYNCSFIK 99 • • •128 TFMLAASWNGTK 139 • • •19 QSPECANLMTVAPITNATMDLLSGK 43 •Table 1: Bovine α1-acid glycoprotein glycopeptides detected by nano LC-MS/MS(Table 1) without any prior enrichment. Only the largest andmost hydrophobic peptides were not detected using any of thephases. Similar results were obtained for bovine fetuin digest.All bovine fetuin glycopeptides could be detected using C 182or C 8 chromatography. However, they were mostly present aslower-charge species, resulting in poorer MS/MS ETD spectrawithout enough information for their unambiguous identification.Previously, Alley and co-workers reported thatfetuin glycopeptides could not be observed after separation ona graphite HPLC Glycan Chip. 11 Our studies indicated thatlarger hydrophobic peptides elute much later in the gradient.Nevertheless, three out of four fetuin peptides were identifiedafter separation on the porous graphite column. Only thelargest O-glycosylated 246-306 peptide was not detected.Figure 5 shows an example of an ETD spectrum of 5+precursor at m/z 1307 of bovine fetuin tri-antennary peptide72 RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR 103 , whichwas successfully identified after separation on porousgraphite column. After optimization, all nano LC-MS/MSanalyses of glycopeptides were performed using a porousgraphite column and 1 pmol of glycoprotein digest.MS /MS Analysis of GlycopeptidesTo identify glycopeptides, we utilized a strategy described byPeterman and Mulholland 2 , where in-source CID generatedcharacteristic oxonium ions at m/z 204 and/or 366 and werefurther fragmented by a dedicated MS 3 event. This allowedfor a highly sensitive and selective detection of the elutingglycosylated peptide ion(s) at a given retention time. Highresolution,accurate-mass full-scan MS was measured in theOrbitrap mass analyzer, as shown in Figure 6, followed bydata-dependent MS/MS alternating CID and ETD scanswith fragments analyzed in either the ion trap or Orbitrapmass analyzers. The resulting CID spectra of the 3+ precursors(Figure 6c) were used for glycan structure elucidationand the ETD spectra of the 4+ precursors (Figure 6b) wereused for glycopeptide identification as demonstrated inFigures 7 and 8 for the bovine α1-acid glycoprotein peptide103 QNGTLSK 109 . Figure 7, inset, shows the CID spectrumof the precursor [M+3H] 3+ at m/z 990.395 measured in theOrbitrap mass analyzer. The oxonium fragment ion atm/z 366 confirmed this precursor as a glycopeptide. Thespectrum was deconvoluted and glycan composition wasassigned as shown in Figure 7. This glycoform contains oneeach of N-acetylneuraminic (Neu5Ac) and N-glycolyeuraminic(Neu5Gc) acids at the glycan branch end as determined bythe presence of 657/673 fragment pair. The product ion atm/z 950.4805 corresponds to [M-GlcNAc+H] + . All measuredmasses were within 5 ppm of their theoretical values whichhelped unambiguously assign the glycan portion as abi-sialated glycopeptide structure.B IOCHEMICALApplication NotesFigure 5: Ion trap MS/MS ETD spectrum of bovine fetuin tri-antennary peptide 72 RPTGEVYDIEIDTLETTCHVLDPTPLANCSVR 103 (5+, m/z 1307). Insert: Magnifiedregion for scan range 100-2000 m/z.13
Page 1 and 2: Applications NotebookIssue 1, June
Page 3 and 4: Applications ReviewApplication Chro
Page 5 and 6: Retention Mechanisms on PGCOn a mol
Page 7 and 8: Porous Graphitic Carbon for the LC/
Page 9 and 10: the retention mechanism is a combin
Page 11: Data ProcessingThermo Scientific Xt
Page 15 and 16: Higher-charged 4+ metal-adducted pr
Page 17 and 18: The Use of Porous Graphitic Carbon
Page 19 and 20: The lowest concentration from the d
Page 21 and 22: The experimental conditions for the
Page 23 and 24: Fast LC Separation of Triazine Herb
Page 25 and 26: SamplesSamples of surface water (Sa
Page 27 and 28: Analysis of Polar Metabolites of At
Page 29 and 30: Compounds Log Kow Recovery (%) S.DA
Page 31 and 32: ExperimentalSample PreparationFor t
Page 33 and 34: Determination of Leucine and its Is
Page 35 and 36: Determination of Occupational Expos
Page 37 and 38: Porous Graphitic Carbon for Inorgan
Page 39 and 40: F OOD AND B EVERAGEFigure 4: Column
Page 41 and 42: Application Area Analyte Group Refe
Page 43 and 44: 69. J. Bessarda, P. Saviuc, Y. Chan
Page 45 and 46: Aflatoxinsafla1Nonylphenol Isomers1
Page 47 and 48: Ordering InformationHypercarb Colum

Thermo Scientific Hypercarb Columns

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