Environmental Engineering Reference
In-Depth Information
since no pores smaller than 30 nm were found in any material and all plasma
proteins investigated had molecular diameters smaller than 15 nm. Thus, steric
exclusion of proteins from the available surface could play a major role in low
protein adsorption onto these surfaces.
15.2.2 Plasma Sampling
The task of collecting large numbers of clinical plasma samples that best represent,
and most accurately reflect, the plasma composition in vivo is of great importance.
Hence accurate plasma processing using standard protocol to yield reliable samples
for proteomic analysis is essential.
Phenyl methyl sulfonyl fluoride (PMSF) and ethylene diamine tetraacetic acid
(EDTA) affect the stability of serum samples [
54
], while Olivieri [
55
] has demon-
strated that the protease inhibitors in red blood cell membrane lysates can have
large effect on 2D-PAGE analysis. For standardised collection and processing of
the plasma samples to obtain reliable and reproducible results via proteomic
analysis, Hulmes et al. have described the role of protease inhibitor cocktail
(PIC) in plasma stability [
56
]. They showed that the inclusion of PIC in the sample
tubes provided stable and reliable human plasma samples that retained stability
under less stringent processing, such as refrigeration for several hours before
processing or one freeze-thaw cycle had little detrimental effect. Samples without
PIC, from either heparin or EDTA plasma tubes, gave results that varied signifi-
cantly from the control samples. Also, even with PIC present in blood tubes, it was
important to quickly decant the separated plasma from the cellular components
found in the blood tubes following centrifugation, as prolonged exposure again
yielded different results from the standard procedure.
15.2.3 Plasma Storage Conditions
Storage temperature also has a profound effect on the protein profile of human
plasma. In a study by Pasella et al. plasma samples were stored for 13 days at
20
C, +4
C and room temperature (20-25
C) prior to proteomic
analysis [
57
]. During storage for a short period (13 days) at four different temper-
atures plasma proteins were more affected by degradation processes at +4
C
compared to the other temperatures studied. However, numerous protein spots
(vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein
A-I, apolipoprotein E, haptoglobin and complement factor B) decreased in abun-
dance with increasing temperature up to 4
C, but at room temperature their
intensity mean values were similar to those of time zero and
80
C,
80
C thus proving
that these proteins are labile at 4
C, but at the same time they are stable at room
temperature (20-25
C). Spots of serum albumin, fibrinogen gamma chain and
