Specific Investigation of Sample Handling Effects on Protease Activities and Absolute Serum Concentrations of Various Putative Peptidome Cancer Biomarkers

Abstract

INTRODUCTION: In the search for novel cancer biomarkers, various proteolytically derived peptides have been proposed to exhibit cancer or cancer-type specificity. As these peptides are presumably also generated after sample collection by tumor-specific proteases, extensive investigation of the involved proteolytic processes is crucial for further research. MATERIALS AND METHODS: Using two previously developed and fully validated liquid-chromatography coupled to tandem-mass spectrometry assays, absolute quantification of, in total, 13 proteolytically derived peptides in human serum was accomplished. The analytes included eight peptides derived from inter-α-trypsin inhibitor heavy chain-4 (ITIH(4)-30, ITIH(4)-29, ITIH(4)-28, ITIH(4)-27, ITIH(4)-26, ITIH(4)-25, ITIH(4)-22, and ITIH(4)-21), bradykinin, des-Arg(9)-bradykinin, Hyp(3)-bradykinin, and fragments from fibrinogen-α-chain (Fib-α ([605-629])) and complement component 4a (C4a ([1337-1350])). Samples were obtained from different healthy individuals and prepared with variable tube types, clotting times, and temperatures. Furthermore, stabilities in the serum fraction were assessed and compared to stabilities in serum from breast cancer patients. RESULTS AND DISCUSSION: The quantitative analyses showed either increasing or decreasing serum concentrations during blood coagulation, while comparable effects were observed in serum separated from the blood clot. Furthermore, comparisons of inter- and intra-individual variations suggested better reflection of an individual's protease activity after prolonged ex vivo incubation. This was illustrated for the putative breast cancer marker ITIH(4)-22, revealing better differentiation after incubation of serum at ambient temperature for 24 h. CONCLUSION: The presented study provides suggestions for more specific and optimized sample preparation, as well as extended knowledge necessary to further explore the opportunities of these proteolytic peptides as cancer biomarkers.

Fig. 1

Effects of clotting time at RT on serum concentrations of a Fib-α _[605–629]; b C4a _{[1337–1350]}; c bradykinin; and d des-Arg⁹-bradykinin in four healthy individuals (represented by different lines). The dotted line represents the assay’s LLOQ

Fig. 2

Effects of clotting time at RT on serum concentrations of a ITIH₄-21; b ITIH₄-22; c ITIH₄-25; d ITIH₄-26; e ITIH₄-27; f ITIH₄-28; g ITIH₄-29 and h ITIH₄-30 in four healthy individuals (represented by different lines). The dotted line represents the assay’s LLOQ

Fig. 3

Effect of clotting temperature on the serum concentrations of the various peptides, expressed as% average response after 24 h clotting at RT compared to 5°C. Error bars indicate the standard deviation (n = 4)

Fig. 4

Measured serum concentrations after blood collection from four healthy individuals (diamonds, squares, triangles, and ex symbols, represented by different lines) in tubes with and without gel separator for the different peptides. Asterisk indicates clotting time of 24 h at 5°C, other samples were allowed to clot for 1 h at RT

Fig. 5

Comparison between effects of incubation time at RT in serum during blood clotting (straight line) and serum after rapid removal of the blood clot (dotted line) on the absolute serum concentrations of a ITIH₄-21 and b ITIH₄-25 in four healthy individuals (diamonds, squares, triangles, and ex symbols)

Fig. 6

Comparison between effects of incubation time at RT in serum during blood clotting (straight line) and serum after rapid removal of the blood clot (dotted line) on the absolute serum concentrations of Fib-α _[605–629] in one healthy individual

Fig. 7

Examples of intra-individual variations (diamonds) in clotting time effects at RT on the serum concentrations of a C4a _{[1337–1350]}, b Des-Arg⁹-bradykinin, c ITIH₄-22 and d ITIH₄-27 in samples collected on three different days at Saltro (dark-gray), UMC (light-gray) or SMCP (black). The dotted line represents the assay’s LLOQ

Fig. 8

Box plots indicating the median ±25th percentile (IQR) for ITIH₄-22 in serum from breast cancer patients (BC, n = 22) and controls (CO, n = 22) after direct measurement, as well as after incubation of the serum for 24 h at RT. The lower and upper bars indicate the lowest and highest value within 1.5 IQR, respectively, while dots represent values between 1.5-3 IQR (outliers) and asterisks values above 3 IQR (extremes)