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4 Notes
- Do not use serum and plasma interchangeably. The clotting
process will remove proteins from serum. Hence, a decision to
use serum or plasma should be made prior to commencing
sample collection. Varying the equilibration buffer on each
protein chip surface will alter the binding of proteins to the
chip surface according to the properties of each protein pres-
ent. The list of buffers in Table 1 can be used in initial screen-
ing experiments to optimize conditions to bind proteins of
interest, for example, by assessing which equilibration buffers
result in the most pronounced differences between patient
sample categories. Selected protein chip and equilibration buf-
fer combinations can be carried forward into assessment of the
full clinical sample set. - Addition of detergent to the equilibration buffer can facilitate
washing of unbound protein. However, detergent can also
bind to the chip surface resulting in anomalous peaks. For this
reason, detergent should not be used with the hydrophobic
H50 chip surface. In addition, very low molecular weight
peaks should be treated with caution. - For serum or plasma samples, try dilutions in the range of 1:10
through 1:100 and assess resultant spectra for resolution
(height and width) of peaks obtained during analysis. This may
depend on the instrument setup being used. - Addition of the matrix is a critical step that will affect the
reproducibility of the spectra obtained during analysis. The
recommended on/off pipetting technique is to depress the
pipette so that the liquid forms a drop at the end of the pipette
tip, then gently lower the pipette so that the drop of liquid
disperses evenly onto the spot, without the pipette tip itself
touching the chip surface. - The detailed information provided in this chapter is based on
settings used on a SELDI-TOF MS PSII instrument. If using
another instrument, different settings may apply. - The laser intensity and detector sensitivity settings are varied in
order to give the best peak heights with lowest background
noise. The optimal conditions may be different when trying to
detect low molecular weight versus high molecular weight pro-
teins. Examples of spectra obtained are illustrated (Fig. 1 ). - The biomarker wizard includes the option to vary the strin-
gency and sensitivity of the analysis. Initially, a more stringent
analysis can be done using for example, a 30% peak threshold
and a signal/noise ratio of 5. Subsequently, where appropriate
a less stringent (more sensitive) analysis can be performed
Peter Kelly