Analysis by 2DE

Samples of albumin and IgG depleted citrate-plasma were subjected to the addition of protease inhibitor cocktail (Fig. 5, Panel A), water (Panel B) or individual protease inhibitor components (Panels C-H), and separated by 2-DE. The presence of the PI cocktail appears to perturb the native pi profiles of several proteins, particularly higher abundance proteins including apolipoprotein A1, apolipoprotein A2, haptoglobin (alpha and beta subunits), transthyretin and fibrinogen - as identified by in-gel tryptic digestion and MALDI-TOF MS (data not shown). Furthermore, the PI cocktail presence induces general smearing of

Fig. 2 The assessment of sample integrity under various storage conditions as assessed by SDS-PAGE, using (A, left panel) 3-8% Tris-Acetate and (A, right panel) 10% Bis-Tris/MOPS. (B) A trace diagram is included showing alterations in selected protein bands with two samples stored at room temperature and liquid nitrogen.

Fig. 2 The assessment of sample integrity under various storage conditions as assessed by SDS-PAGE, using (A, left panel) 3-8% Tris-Acetate and (A, right panel) 10% Bis-Tris/MOPS. (B) A trace diagram is included showing alterations in selected protein bands with two samples stored at room temperature and liquid nitrogen.

the entire 2-DE protein profile, and 13% fewer resolved spots, although it is possible that addition or alteration of sample preparation steps could eliminate at least the smearing.

The changes in the isoform profiles are exemplified by apolipoprotein A1 (Fig. 5, boxes in panels) in which the intensity of the two high pi isoforms are substantially increased with the inclusion of the PI cocktail. The relative intensities of the protein spots from panels A and B were compared using the 2-DE gel analysis software. The intensities of the lower pi isoform spots decrease with PI added, and there is a concomitant increase in the intensities ofthe higher pI isoforms (data not shown).

Tab. 4 Changes in SELDI-TOF profiles of serum samples under different storage conditions. Samples were analyzed in duplicate and alterations in peak intensities were noted after the generated profiles were analyzed using Ciphergen's Biomarker Wizard™1. The presence of a peak is denoted by a check mark, whereas the absence is denoted by an X. The control sample corresponds to a sample that was prepared, aliquoted and denatured at t = 0 (see Section 2 for details) and serves as a basis for comparison for the other timed samples

m/z

Control

23 °C

23 °C

4°C

4°C

20°C

20°C

80°C

80°C

Liquid

Liquid

Notes

(t = 0)

8h

7d

2d

7d

3 Od

60d

30d

eod

nitrogen 30d

nitrogen 60d

3900

Specific to 23°C

4100

Specific to 23°C and 4°C

7200

Specific to 23°C

7600

Undetected at 23°C

13600

Specific to 23°C and 4°C

14500

Specific to 23°C

17300

Undetected at 23°C

28000

Undetected at 23°C

90000

Specific to 23°C

95000

Undetected at 23°C

Fig. 3 2-D analysis of sample storage conditions. (A) Human serum stored at extreme low (LN2, left) and high (230C, right) temperature conditions. 100 mg serum was separated on linear pH 3-10 IPG strip (first dimension) 2-D gels and stained with SYPRO Ruby. Several representative areas where protein spots appear to be

Fig. 3 2-D analysis of sample storage conditions. (A) Human serum stored at extreme low (LN2, left) and high (230C, right) temperature conditions. 100 mg serum was separated on linear pH 3-10 IPG strip (first dimension) 2-D gels and stained with SYPRO Ruby. Several representative areas where protein spots appear to be missing, or changing in intensity have been highlighted. (B) Enlargements of areas highlighted in panel A are shown for all temperature conditions. These enlargements illustrate the disappearance of some protein spots at higher temperatures and the appearance of new spots indicating proteolytic fragments.

In order to determine which PI component(s) were responsible for this observed effect, citrate-plasma samples were subjected to each individual protease inhibitor at the same concentration as is present in the cocktail, using otherwise identical methods as above. Gels were compared for each ofthe six protease inhibitor treated samples. The gel in which 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was added shows similar perturbations of native patterns to those seen using the complete protease inhibitor cocktail, (Fig. 5, Panels A and C). Samples run in the presence of the other protease inhibitors show sharply resolved spots and the same isoform profiles as seen without the inhibitor cocktail.

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