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Methodentexte zum Einfügen in Publikationen und Abschlussarbeiten

Bestimmung der intakten Proteinmasse, denaturierend:

Depending on their concentration and the expected protein masses, 1-10 µL of the buffered protein solutions were desalted online using a Waters ACQUITY H-Class HPLC-system equipped with a MassPrep column (Waters). Desalted proteins were eluted into the ESI source of a Synapt G2Si mass spectrometer (Waters) by the following gradient of buffer A (water/0.05% formic acid) and buffer B (acetonitrile/0.045% formic acid) at a column temperature of 60°C and a flow rate of 0.1 mL/min: Isocratic elution with 5% A for two minutes, followed by a linear gradient to 95% B within 8 minutes and holding 95% B for additional 4 minutes.

Positive ions within the mass range of 500-5000 m/z were detected. Glu-Fibrinopeptide B was measured every 45s for automatic mass drift correction. Averaged spectra were deconvoluted after baseline subtraction and eventually smoothing using MassLynx instrument software with MaxEnt1 extension.

Bestimmung der intakten Proteinmasse, nativ:

Probenvorbereitung (Entsalzung/Umpufferung auf Ammoniumacetat) auf Kundenseite.

Für die eigentliche massenspektrometrische Analyse:

Samples were infused through the instruments internal sample syringe (line A). Positive ions within the mass range of 500-12.000 m/z were detected. Data were acquired in manual mode without automatic mass drift correction. Averaged spectra were deconvoluted after baseline subtraction and eventually smoothing using MassLynx instrument software with MaxEnt1 extension.

Der Massenbereich kann für größere Komplexe von den angegebenen 500-12.000 m/z abweichen falls Signale oberhalb von 12.000 liegen sollten. 

Protein-Identifizierung/Proteomics:

Da wir aktuell zwei unterschiedllche Geräte für Proteomics betreiben und je nach Art der Proben auch unterschiedliche Probenvorbereitungen durchführen fragen Sie bei Bedarf die individuellen Texte bitte an.