Preanalytic influence of sample handling on SELDI-TOF serum protein profiles.
BACKGROUND
High-throughput proteomic methods for disease biomarker discovery in human serum are promising, but concerns exist regarding reproducibility of results and variability introduced by sample handling. This study investigated the influence of different preanalytic handling methods on surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) protein profiles of prefractionated serum. We investigated whether older collections with longer sample transit times yield useful protein profiles, and sought to establish the most feasible collection methods for future clinical proteomic studies.


METHODS
To examine the effect of tube type, clotting time, transport/incubation time, temperature, and storage method on protein profiles, we used 6 different handling methods to collect sera from 25 healthy volunteers. We used a high-throughput, prefractionation strategy to generate anion-exchange fractions and examined their protein profiles on CM10, IMAC30-Cu, and H50 arrays by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.


RESULTS
Prolonged transport and incubation at room temperature generated low mass peaks, resulting in distinctions among the protocols. The most and least stringent methods gave the lowest overall peak variances, indicating that proteolysis in the latter may have been nearly complete. For samples transported on ice there was little effect of clotting time, storage method, or transit time. Certain proteins (TTR, ApoCI, and transferrin) were unaffected by handling, but others (ITIH4 and hemoglobin beta) displayed significant variability.


CONCLUSIONS
Changes in preanalytical handling variables affect profiles of serum proteins, including proposed disease biomarkers. Proteomic analysis of samples from serum banks collected using less stringent protocols is applicable if all samples are handled identically.
Timms
John F
JF
Translational Research Laboratory, Institute of Women's Health, University College London, London, United Kingdom. jtimms@ludwig.ucl.ac.uk
Arslan-Low
Elif
E
Gentry-Maharaj
Aleksandra
A
Luo
Zhiyuan
Z
T'Jampens
Davy
D
Podust
Vladimir N
VN
Ford
Jeremy
J
Fung
Eric T
ET
Gammerman
Alex
A
Jacobs
Ian
I
Menon
Usha
U
D001798:Blood Proteins / Q000032:analysis*; D001800:Blood Specimen Collection / Q000379:methods*; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D017698:Postmenopause; D040901:Proteomics; D016032:Randomized Controlled Trials as Topic; D044967:Serum; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001798:Blood Proteins