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Letters to the Editor |
1 Institute for Clinical Chemistry 2 Department of Surgery and 3 Department of Radiation Oncology, Medical Faculty Mannheim of the University of Heidelberg, Mannheim, Germany
aAddress correspondence to this author at: Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, Theodor Kutzer Ufer 1-3, 68167 Mannheim, Germany. Fax 49-621-383-3819; e-mail peter.findeisen{at}ikc.ma.uni-heidelberg.de.
To the Editor:
Serum has proven a difficult matrix for mass spectrometry (MS)-based clinical proteomic profiling. Problems occur mainly as a result of preanalytical variables in sample handling and processing, causing substantial changes in MS peptide profiles (1) that can completely abolish meaningful data interpretation(2). The necessary rigorous standardization of sample collection and processing procedures is difficult to integrate into routine laboratory testing. Furthermore, no new biomarker proteins have emerged from profiling experiments of serum specimens; thus far only proteolytic fragments of high-abundance serum proteins have been reported as markers(3). Proteases shape the peptide pattern of serum specimens in a time-dependent manner(1), and disease-related proteases seem to generate a characteristic pattern of proteolytic fragments from abundant endogenous proteins(4).
We hypothesized that addition of exogenous reporter peptides (RPs) to serum will also enable characterization of protease activity in serum samples while offering a major advantage: The standardization of RP addition with respect to incubation time, temperature, and substrate concentration is quite simple and might alleviate the preanalytical difficulties of current profiling approaches.
To test this hypothesis we generated an RP mixture by tryptic digestion of a randomly chosen recombinant protein. The N-terminally 6xHis-tagged fragment of the adenomatous polyposis coli protein that covers amino acids 3–312 (NP_000029) was expressed using the pQE30 vector (Qiagen) and Escherichia coli XL1-Blue cells. Protein was purified using Ni-Sepharose before carbamidomethylation and in-solution digestion with trypsin (Promega). The resulting peptides were purified using OmixTM C18 pipette tips (Varian), then organic solvent of the eluate was evaporated and peptides were reconstituted with PBS, pH 7.4 (cat. no. H15-002; PAA Laboratories), to yield the final RP mixture solution.
We mixed 30 µL of serum and 20 µL of the RP mixture and incubated the mixture at 37 °C for 2 h. In control samples, we substituted 20 µL PBS for the RP mixture. Before MS (MALDI-TOF Autoflex II, Bruker) the samples were processed using magnetic bead-based hydrophobic interaction chromatography on C18 resins (Bruker). Blood was collected from 50 healthy employees of the University Hospital Mannheim during routine laboratory testing at the employee medical facility. All specimen donors gave informed consent, and the local ethics committee approved the study.
Serum was centrifuged at 20 °C for 10 min at 3000g and stored at –80 °C. To investigate preanalytical impact on MS profiles, the serum of 1 donor was divided into aliquots and stored at room temperature for 1–6 h before freezing at –80 °C. For measurements, all aliquots were thawed once and processed in parallel with or without the addition of the RPs (Fig. 1A
).
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The MS patterns of peptide-supplemented sera were comparable among samples from healthy individuals. Within-day and between-day reproducibility of MS-spectra were assessed as previously described (1), and the coefficient of determination (R2) in any case was 
0.93 (data not shown), confirming good reproducibility of magnetic bead-based sample processing.
Incubation of RPs with serum changed the peptide profile. By comparing samples before and after incubation for 2 h at room temperature, we distinguished the following peak categories (Fig. 1B
): Peaks designated category A originated from serum. Peaks designated category B were RPs of the RP mixture that were not found in the sera after addition of the RP mixture, suggesting that they either were rapidly degraded by serum proteases or were quenched owing to ion suppression. Peptides designated category C originated from the RP mixture but with varying signal intensities. Peaks designated category D appeared de novo in serum as a result of proteolytic cleavage of RP peptides by endogenous proteases.
We designated 2 different preanalytical periods, periods I and II, as outlined in Fig. 1A
. As exemplified in Fig. 1B
, different extensions of preanalytical period I resulted in distinct changes of MS-peptide profiles and a time-dependent decrease of the m/z 1466 category A peak (1). In contrast, category D peaks (m/z: 1364, 1741, and 1899; Fig. 1B
) that appeared in enriched sera did notshow differences in signal intensity despite variability in preanalytical period I, demonstrating that protease activity was unaffected over prolonged periods of sample storage.
In conclusion, these preliminary results demonstrate that addition of RP to serum specimens can alleviate preanalytical variability. The feasibility of RP addition has already been demonstrated for diagnosis of thrombotic thrombocytopenic purpura associated with altered activity of a single protease (5). A future challenge will be the composition of an optimized RP mixture for MS-based protease profiling of other conditions such as malignancies that are also associated with specific protease patterns.
Acknowledgments
Grant/funding support: This project was supported in part by a grant from the Deutsche Krebshilfe (project No. 106856) and the Lesser-Loewe Foundation e.V.
Financial disclosures: None declared.
References
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