Page 51 - Macromolecular Crystallography

40 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 2.11 Automated desalting protocol for ESI-MS analysis of intact proteins

1. Samples are diluted to ∼10 µM in a 96-well PCR plate. 4. The protein is then eluted to ESI-MS with 20% water/
2. The protein is injected onto a 1 cm × 5 µmC4 80% acetonitrile containing 0.1% formic acid.
precolumn (Anachem) using an autosampler. 5. Simple MS measurements are taken and the raw data
3. The column is washed to waste with water containing deconvoluted to give an accurate mass of the intact protein
2% MeOH, 0.5% formic acid, and 0.2% trifluoroacetic acid, sample.
thus removing salts from the sample.

Table 2.3 Routine methods for quality assessment undertaken in the diameter can be used to estimate the total molecular
Oxford Protein Production Facility
weight, and thus the oligomeric state of the pro-
tein present. If more than one oligomeric state exists
Quality assurance technique Protein characteristic
within a sample, such heterogeneity can reduce the
SDS-PAGE Purity and denatured molecular crystallizability of the sample (Habel et al., 2001).
weight DLS is therefore a simple, fast measure of the multi-
UV spectrometry Protein concentration meric state and monodispersity of a protein sample.
Protein assay (BioRad) Protein concentration
DLS techniques have been taken further with the
Size exclusion chromatography Purity and oligomeric state
introduction of 96- and 384-well format equipment
Dynamic light scattering Polydispersity and oligomeric
such as the DynaPro™ plate reader from Wyatt
state
Technology.
LC-ESI-MS Accurate denatured molecular
weight, purity, and bound
ligands
2.5 Summary and future perspectives
A major driving force for the development of
sequencing of regions of the protein, although full
high-throughput cloning, expression, and pro-
sequence coverage is rare in this type of experiment
tein purification has been the arrival of the post-
(Cohen and Chait, 2001). In addition, information
genomic era, where the emphasis has changed
regarding post-translational modifications such as
from DNA sequencing to structural (and functional)
phosphorylation and glycosylation can be attained
proteomics. A wide variety of HTP methodolo-
using MS.
gies have been successfully implemented in major
world-wide efforts to generate protein for crystallo-
2.4.5.2 Light scattering graphic and NMR structure determination. For the
Dynamic light scattering (DLS) is used to deter- development of HTP protein production pipelines,
mine the oligomeric state and monodispersity of a many of the initial projects have focused on bac-
protein. A coherent, monochromatic beam of light terial genomes, where high-throughput production
is passed through a protein sample in solution and of proteins expressed solubly in E. coli is read-
the particles scatter the light in all directions. As the ily achieved. In the future, the emphasis will
particles move according to Brownian motion, they shift increasingly to eukaryotic and viral genomes
cause time-dependent fluctuations in the scattering where production of soluble proteins remains a key
of the light. By measuring the time-dependence of issue. To what extent will current methodologies be
the fluctuations, the diffusion coefficient of the par- adequate for tackling structural proteomics in the
ticles can be calculated. From the Stokes–Einstein future? Clearly, ligation-independent cloning meth-
equation, the viscosity of the medium and this dif- ods have been a major success in providing com-
fusion coefficient, the diameter of the particles can pletely generic protocols that are readily automated.
be calculated. Using a globular model, the particle The inherent difficulties of expressing eukaryotic