Zeba spin column

Prior to MS analysis, samples were desalted using 0.5 ml Zeba™ Spin Columns (Life Technologies, Pierce Biotechnology, Rockford, IL, USA) in 200 mM ammonium acetate pH 7.5.
For non-denaturing MS analysis, samples were directly injected after desalting, whereas denaturing conditions were obtained by an additional 2-fold dilution with 50% acetonitrile (ACN), 0.1% formic acid. In both cases, analyses were performed on a LCT (Waters, Manchester, UK) coupled to an automated chip-based nano-electrospray source (Triversa Nanomate, Advion Biosciences, Ithaca, NY, USA) and running in positive ion mode. The instrument parameters in denaturing conditions were as follow: cone voltage was set to 30 V, with an interface pressure of 1.5 mbar. In non-denaturing MS conditions, the cone voltage was increased to 120 V and the interface pressure to 6 mbar. The instrument was calibrated with a 2 μM solution of horse heart myoglobin (Sigma-Aldrich) for denaturing MS and with a 2 mg/ml cesium iodide solution in 50% isopropanol for non-denaturing MS. Data analysis was performed with MassLynx 4.1 (Waters, Manchester, UK).

First, 10 nmol of hexahistidine-GST-Rab was loaded with 2′-(3′)-bis-O-(N-methylanthraniloyl)-GDP (Mant-GDP; Jena Bioscience) in 20 mM Hepes, pH 6.8, 1 mg/ml BSA, 20 mM EDTA, pH 8.0, and 40 mM Mant-GDP at 30°C for 30 min. After loading, 25 mmol MgCl₂ was added and the sample was exchanged into reaction buffer (20 mM Hepes, pH 6.8, 1 mg/ml BSA, 150 mM NaCl, and 1 mM MgCl₂) using Zeba spin columns (Thermo Fisher Scientific). Nucleotide exchange was measured using 1 nmol of the loaded Rab and the amount of GEF was specified in the figure legends in a final volume of 100 µl reaction buffer by monitoring the quenching of fluorescence after release of Mant-GDP using a Tristar LB 941 plate reader (Berthold Technologies) under control of MikroWin software. Samples were excited at 350 nm and emission monitored at 440 nm. GTP was added to a final concentration of 0.1 mM to start the exchange reaction at 30°C. Curve fitting and extraction of pseudo first-order rate constants (k_obs) was performed as described previously (Delprato et al., 2004 (link); Delprato and Lambright, 2007 (link)). Because k_obs = (k_cat/K_m) [GEF] + k_basal, where k_basal is the rate constant measured in the absence of GEF, catalytic efficiency (k_cat/K_m) can be obtained.

The primary sample 8670 NISTmAb (PS 8670,
an in-house primary standard material), lot 3f1b, is an IgG1k mAb
derived from a separate production lot of NISTmAb 8671 expressed in
murine suspension culture.⁴ It was obtained
from the Bioanalytical Science Group at NIST. Digestion reagents guanidine
hydrochloride, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine
(TCEP), N-ethylmaleimide (NEM), and iodoacetamide
(IAM) were purchased from Sigma-Aldrich (St. Louis, MO). A revised
Tris buffer (pH = 7) was prepared via adjustment of tris base solution
(200 mmol) by HCl solution (1 mol/L). Sequencing-grade trypsin was
purchased from Promega (Madison, WI). RapiGest, brand-name for sodium
3-[(2methyl-2-undecyl-1,3-dioxolan-4-yl)methoxy]-1-propanesulfonate,
was purchased from Waters (Milford, MA); Zeba spin columns (7 K molecular
weight cutoff (MWCO)) were purchased from Thermo Fisher Scientific
(Waltham, MA). Chromatographic separations were performed on an Acclaim
pepmap100 nano column (150 mm × 75 μm, C18, 3 μm
particle size, 100 Å pore size, Dionex, Sunnyvale, CA).