title: Spiking of serum specimens with exogenous reporter peptides for mass spectrometry based protease profiling as diagnostic tool.
abstract: Serum is a difficult matrix for the identification of biomarkers by mass spectrometry (MS). This is due to high-abundance proteins and their complex processing by a multitude of endogenous proteases making rigorous standardisation difficult. Here, we have investigated the use of defined exogenous reporter peptides as substrates for disease-specific proteases with respect to improved standardisation and disease classification accuracy. A recombinant N-terminal fragment of the Adenomatous Polyposis Coli (APC) protein was digested with trypsin to yield a peptide mixture for subsequent Reporter Peptide Spiking (RPS) of serum. Different preanalytical handling of serum samples was simulated by storage of serum samples for up to 6 h at ambient temperature, followed by RPS, further incubation under standardised conditions and testing for stability of protease-generated MS profiles. To demonstrate the superior classification accuracy achieved by RPS, a pilot profiling experiment was performed using serum specimens from pancreatic cancer patients (n = 50) and healthy controls (n = 50). After RPS six different peak categories could be defined, two of which (categories C and D) are modulated by endogenous proteases. These latter are relevant for improved classification accuracy as shown by enhanced disease-specific classification from 78% to 87% in unspiked and spiked samples, respectively. Peaks of these categories presented with unchanged signal intensities regardless of preanalytical conditions. The use of RPS generally improved the signal intensities of protease-generated peptide peaks. RPS circumvents preanalytical variabilities and improves classification accuracies. Our approach will be helpful to introduce MS-based proteomic profiling into routine laboratory testing.
authors.affiliation: Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Germany. peter.findeisen@ikc.ma.uni-heidelberg.de
authors.forename: Peter
authors.initials: P
authors.lastname: Findeisen
authors.forename: Teresa
authors.initials: T
authors.lastname: Peccerella
authors.forename: Stefan
authors.initials: S
authors.lastname: Post
authors.forename: Frederik
authors.initials: F
authors.lastname: Wenz
authors.forename: Michael
authors.initials: M
authors.lastname: Neumaier
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000737:chemistry; D015415:Biomarkers / Q000097:blood; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D010190:Pancreatic Neoplasms / Q000097:blood / Q000175:diagnosis; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry; D010447:Peptide Hydrolases / Q000097:blood*; D040081:Protein Array Analysis; D040901:Proteomics; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D015415:Biomarkers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010447:Peptide Hydrolases