title: Pre-analytical operating procedures for serum Low Molecular Weight protein profiling.
abstract: Biological specimen collection and storage are an integral component of serum proteomics research. Although many efforts have been posed to address the effects of pre-analytical procedures, standardized protocols for collection and storage of samples for Low Molecular Weight (LMW) proteome profiling are still needed. Here we report a systematic analysis on the influence of pre-analytical factors [clotting times, temperature and time storage, addition of protease inhibitor (PI)] on serum LMW proteome profiling. Moreover, a comparison between manual versus automated peptide purification by functionalized magnetic bead-based MALDI-MS approach was performed. The results demonstrated best serum LMW proteins recovery and stability using a clotting time between 1 and 2h, with serum stored up to 2h either at room temperature or at 4 degrees C, independently of PI addition. PI addition to whole blood resulted in a lower number of LMW peaks detected. Finally, minimal effects on serum proteome profiles were observed after 1-month storage at -80 degrees C, independently of PI addition on whole blood and/or serum. In conclusion, the use of standardized pre-analytical and storage procedures together with an automated peptide purification might minimize potential bias on serum LMW profiling results, thus allowing a better homogeneity and reproducibility in future proteomics studies.
authors.affiliation: Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana, Rome, Italy. francesco.digirolamo@sanraffaele.it
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Girolamo
authors.forename: Jhessica
authors.initials: J
authors.lastname: Alessandroni
authors.forename: Paolo
authors.initials: P
authors.lastname: Somma
authors.forename: Fiorella
authors.initials: F
authors.lastname: Guadagni
mesh_terms: D000328:Adult; D001777:Blood Coagulation / Q000502:physiology; D001779:Blood Coagulation Factors / Q000502:physiology; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001800:Blood Specimen Collection / Q000379:methods; D019411:Clinical Laboratory Techniques / Q000295:instrumentation; D005260:Female; D006461:Hemolysis / Q000502:physiology; D006801:Humans; D008297:Male; D055442:Metabolome; D008863:Microspheres; D008970:Molecular Weight; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D055815:Young Adult
chemical_list: D001779:Blood Coagulation Factors; D001798:Blood Proteins; D020543:Proteome