Jos Jansen

34 Chapter 2 Nanochip-C8 QTOF Mass Spectrometry of Intact Tf For high-resolution mass spectrometry of the intact Tf protein, 10 μ l of serum sample was incubated with anti-Tf beads before injection, and the eluate was analyzed on a microfluidic nanoLC-C8-chip 6540 QTOF instrument (Agilent Technologies).(14) Agilent Mass Hunter Qualitative Analysis Software B.04.00 was used for data analysis. For deconvolution of the charge distribution raw data, Agilent BioConfirm Software was used. Metabolic Labeling with Alkyne-Tagged Modified Sugar Metabolic labeling was performed as described before.(23) Primary skin fibroblasts were maintained in DMEM supplemented with 10% fetal bovine serum (Lonza), at 37°C under 5% CO2 atmosphere. Fibroblasts were grown overnight on glass coverslips (12 mm diameter). Medium was then changed with pre-warmed medium containing 500 mM of alkynyl-modified sugar (ManNAl, provided by Dr. Y. Geurardel and Prof. C. Biot). Labeling lasted 8 hr or 6 hr. The labeling was stopped by fixing the cells with 4% PFA. Cells were then permeabilized in 0.5% Triton X-100 for 10 min. After washes, cells were incubated in the click chemistry buffer containing CuSO4, 5H2O-BTTAA- ascorbate-potassium phosphate, and azide-fluor 545 (Sigma, #760757). The pool of fluorescent glycoconjugates was visualized through an inverted Leica TCS-SP5 confocal microscope. Pictures were taken with the Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems). For comparison purposes, each picture was taken under the same settings. TISGolgi was used to automatically detect the Golgi area and measure the Golgi fluorescence. This homemade ImageJ plugin was developed by TISBio (see Web Resources). Three different fields of two independent experiments were examined.