Specific Investigation of Sample Handling Effects on Protease Activities and Absolute Serum Concentrations of Various Putative Peptidome Cancer Biomarkers
© The Author(s) 2010
Published: 30 September 2010
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 (ITIH4-30, ITIH4-29, ITIH4-28, ITIH4-27, ITIH4-26, ITIH4-25, ITIH4-22, and ITIH4-21), bradykinin, des-Arg9-bradykinin, Hyp3-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 ITIH4-22, revealing better differentiation after incubation of serum at ambient temperature for 24 h.
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.
Mass spectrometry-based profiling of the serum proteome is a well-known approach in the search for biomarker proteins able to predict specific cancer types in an early stage [1, 2]. As a result, also the low molecular weight serum proteome, or peptidome, has attracted increasing attention [3–5], and this has resulted in the proposal of various peptides with putative diagnostic characteristics for different types of cancer in the past years [6–11]. In addition to the classical principle that altered protein levels are directly caused by the cancer type, the upregulated or downregulated peptide concentrations have been related to cancer-specific interferences with protein or peptide breakdown pathways [3, 5]. Moreover, tumor-specific proteases, including endo- and exopeptidases, have been postulated to contribute to different serum peptidome profiles, also suggesting ex vivo generation of candidate discriminatory peptides after blood collection [10, 12, 13].
This hypothesis has received much criticism. On the one hand, because it is likely to be highly subjected to uncontrollable variables during sample collection, and on the other hand, because of the questionability of the tumor’s influence on the ex vivo peptide generation. As this process depends on the activity of coagulation and complement cascades, influences of other factors such as coagulation disturbances or infections are suggested [14–16].
The contribution of various non-cancer-related factors to serum peptidome profiles is widely accepted as a major source of bias and one generally agrees on strict adherence to completely standardized procedures [17–19]. Several studies on the influence of pre-analytical factors on the serum peptidome profile have been performed to further optimize the standardized protocols. Especially the type of blood collection tube, variations in clotting time and temperature, storage conditions, and amount of freeze-thaw cycles have been investigated and shown to have significant effects on the serum peptidome profile [18–24]. For example, effects of clotting time have been categorized by Umemura et al.  as types A, B, or C, in which types A and B indicate decreasing and increasing serum concentrations, respectively, with increased clotting time. Type C is defined as showing an initial increase, while remaining relatively stable thereafter.
However, the abovementioned studies on sample handling effects were not quantitative and based on general observations of spectral changes, not providing any specific information on particular peptides. Therefore, to better understand the effects of sample handling variables on the specific expression in serum of putative peptidome markers generated after blood collection, novel quantitative methods will be beneficial.
Amino acid sequences of the investigated proteolytic peptides
Amino acid sequence
Now, with the use of these quantitative methods, we specifically investigated the effects of clotting time, the blood collection tube, and clotting temperature on the serum concentrations and stabilities of these peptides. Furthermore, the effects of ex vivo protease activities in serum were compared between several breast cancer patients and controls. Expanded knowledge on the abovementioned effects is required to further explore the biomarker potential of the different peptides for early-cancer diagnosis. For example, little differences in sample handling can be a possible explanation for the observed differences between the study of Villanueva et al.  and the recently reported quantitative results [29, 30]. The specific investigations described here can help in optimizing sample handling protocols for each particular peptide, ideally resulting in (1) the lowest possibility of confounding factors to affect the serum peptide concentration; (2) the highest expression of the peptide formed after proteolytic cleavages; and (3) the most significant differentiation between protease activities in serum from controls and cancer patients.
Materials and Methods
Blood Collection and Serum Preparation
Blood was collected at three different locations. At Saltro Diagnostic Center Utrecht (Utrecht, The Netherlands), blood was collected from four healthy individuals in micronized silica-coated plastic (polyethylene terephthalate; PET) tubes from Greiner Bio-One (Alphen a/d Rijn, The Netherlands), either with (4 ml #454071) or without (4 ml #454314) an inert olefinoligomer barrier gel. From one of these four individuals, additional blood samples were taken at two other locations with time intervals of ca. 1 month between each occasion of blood collection. In the Utrecht Medical Center (UMC, Utrecht, The Netherlands), plastic (PET) silicone-coated tubes with clot activator (micronized silica particles) and a polymer gel separator from Becton Dickinson (Breda, The Netherlands) were used (Vacutainer 3.5 ml SST tubes, #367057). At the third location, Sports Medical Center Papendal (SMCP, Arnhem, The Netherlands), blood was collected in the same plastic tubes with clot activator and gel separator as used at Saltro (Greiner Bio-One; 4 ml #454071).
In all cases, blood of each volunteer was collected in seven tubes that contained clot activator and gel separator and were thereafter allowed to clot at room temperature (RT) for 0.5, 1, 2, 4, 6, 8, or 24 h. From the four individuals, one additional tube with gel separator was allowed to clot for 24 h at 5°C, while the two plastic tubes without gel separator were kept either at RT for 1 h or at 5°C for 24 h.
After the specified clotting time, all samples were centrifuged at 1,500×g for 10 min at 23°C. Subsequently, three 100-μl aliquots of serum were taken, while the remaining serum was divided into 500-μl portions. All aliquots were immediately stored at −80°C after preparation.
All samples were thawed and further processed on ice. Aliquots of 500 and 100 μl were purified by solid-phase extraction on mixed-mode weak cation exchange  or silica C2 reversed-phase sorbents , respectively.
For assessing the stabilities in serum, 100 μl aliquots from the sample with the shortest clotting time (30 min) were left at RT for 0.5, 1.5, 3.5, 5.5, 7.5, and 23.5 h. This was also performed with 500-μl aliquots of the blood collected at the SMCP from one individual. Furthermore, for all individuals, additional serum aliquots were left at RT for 4 h, using two serum samples from both tube types, with clotting procedures of either 1 h at RT or 24 h at 5°C.
The LC-MS/MS analyses were performed as described in detail previously [27, 28]. Both assays were performed using an Accela high-speed chromatographic system coupled to a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with a heated electrospray ionization source (all from Thermo Fischer Scientific, San Jose, CA, USA), using multiple reaction monitoring for detection.
For both procedures, calibration standards and quality control (QC) samples were prepared in analyte-free bovine plasma, using stable-isotope labeled peptides or structural analogs as internal standards. Accuracies and precisions of the QC samples were determined to monitor analytical performance, and the quantitative analysis was accepted when deviations from the expected concentrations and variations were below 15%.
Protease Activities in Serum from Breast Cancer Patients
Serum aliquots of 500 μl from nine breast cancer patients, as well as from nine controls were incubated at RT for 24 h, subsequently analyzed by LC-MS/MS to quantify serum levels of the six putative breast cancer marker peptides. Furthermore, 100 μl aliquots from 13 different patients and controls were prepared in duplicate to obtain absolute serum concentrations of the eight ITIH4-derived peptides: one aliquot was directly further processed, while the other sample was left at RT for 24 h before further processing.
All serum samples were obtained uniformly following a strict standardized protocol at two different locations: the Slotervaart Hospital and the Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital (both in Amsterdam, The Netherlands) after approval by the local medical ethics committees of both hospitals, and after receiving individuals’ written informed consent. The serum samples from the breast cancer patients were obtained prior to surgery, and none of them was undergoing treatment. Most breast cancer patients were postmenopausal except for five premenopausal and two menopausal women. The serum samples from the controls (median age 56.1 years; inter-quartile range (IQR) 46.4–69.5 years) were matched to the serum samples from the breast cancer patients for age (median age 56.2 years; IQR 44.7–69.9 years) and storage duration (median 41 months for both groups).
Blood was collected in 9.5 ml BD Vacutainer® SST™ tubes (Beckton-Dickinson) and allowed to clot for exactly 30 min at RT, after which they were centrifuged at 1,500×g for 15 min at RT and subsequently aliquoted and stored at −80°C.
Results and Discussion
Effect of Clotting Time
The concentrations of C4a [1337–1350] were low in all serum samples (max. 2.3 ng/ml) and below the lower limit of quantification (LLOQ) for several samples (Fig. 1b). Nevertheless, a small initial increase in the serum concentration could be observed during the first 2 h of clotting, thereafter slightly decreasing and this type of effect was classified as type C . This effect probably indicates a reduced amount of the founder peptide, i.e., complement component 4a, with C4a [1337–1350] as an intermediate in further proteolytic processing. Besides the formation of proteolytic cascades, blood clotting can stimulate complement activation, although the exact mechanism of this interplay remains unclear . On the other hand, activation of the complement cascade by blood coagulation can explain the initial increase in the proteolytic fragment of C4a, which is thereafter susceptible to consecutive proteolytic cleavages.
Bradykinin, des-Arg9-bradykinin, and Hyp3-bradykinin showed rapid decreases with prolonged clotting time (Fig. 1c, d), and maximum serum concentrations were measured in the sample with the shortest clotting time (type A ). However, for des-Arg9-bradykinin, a C-type initial increase during the first 30 min can be expected due to proteolytic cleavage of bradykinin. Bradykinin is a key component in the intrinsic pathway of coagulation as the first step is activation of kallikrein, a serine proteinase which cleaves high molecular weight kininogen and releases bradykinin [31, 33]. However, bradykinin is also known to be rapidly degraded ex vivo, for example by carboxypeptidases N at the 8–9 position to yield des-Arg9-bradykinin. Both bradykinin and des-Arg9-bradykinin are predominantly cleaved by angiotensin I converting enzyme at the 7–8 and 5–6 positions . The type-A effect for both peptides indicates that bradykinin is generated at the beginning of the ex vivo intrinsic pathway  and then rapidly degraded to mainly des-Arg9-bradykinin which is thereafter highly susceptible to further proteolytic cleavages.
Although these results show that blood coagulation affects the serum concentrations of all peptides, they cannot be solely attributed to the clotting process. Thorough investigation of the specific role of the involved proteases and the founder peptides as well as their relation with blood coagulation and fibrinolysis is, therefore, required. For example, it remains unclear whether the different extent of the observed effects between the four individuals is caused by the presence of different proteases or by differences in activities of the same combination of proteolytic enzymes.
On the other hand, these results indicate the relevance of adherence to strict standardized protocols as the ongoing proteolytic breakdown in blood during the clotting process easily introduces pre-analytical variance. Furthermore, the protease activities can have completely opposite effects on the various peptides, apparently necessitating different blood collection procedures for the different peptide types. For example, adequate quantification of the ITIH4 peptide fragments in clinical serum samples, with clotting times of exactly 30 min, was seriously complicated by the low expression of these peptides [26, 29]. Prolonged clotting time of the blood samples could be beneficial for more accurate analytical measurements. Contrary, analysis of bradykinin and Hyp3-bradykinin likely requires very short clotting times, although their ex vivo degradation occurs so rapidly that robust serum preparation will still be seriously complicated. Interestingly, Yi et al.  suggest the addition of protease inhibitors prior to blood collection to reduce artificial generation of bradykinin by activation of the kallikrein–kinin system by surface contact with blood collection devices. Nevertheless, as long as the tumor’s effect on the endo- and/or exoprotease activity, and thus eventually on the bradykinin serum concentrations, is not elucidated, the most appropriate sample preparation procedures remain speculative.
Effect of Clotting Temperature
With the observed influence of clotting temperature on the final serum concentrations, careful control and registration of the clotting temperature are required. Additionally, clotting at lowered temperatures can be beneficial to minimize the effects of small variations in clotting time and/or to provide higher and more accurately measurable concentrations for specific peptides as observed for C4a [1337–1350] and likely expected for bradykinin peptides.
Effect of Collection Tube
For most peptides, serum concentrations were higher if prepared in tubes with gel separator (Fig. 4). Only for A-type peptides, with observed ex vivo degradation, lower concentrations were measured, suggesting increased protease activity by the gel. However, C4a [1337–1350], des-Arg9-bradykinin, ITIH4-21, and ITIH4-28 showed some contrasting results for at least one individual. For C4a [1337–1350], this could possibly be attributed to the absence of the gel barrier between serum and blood clot after centrifugation in one of the tubes (▲). Des-Arg9-bradykinin on the other hand showed contrasting effects for the samples with a low concentration in the tubes with gel, compared to the samples with a clearly higher concentration, due to higher bradykinin concentrations in these individuals (Fig. 1). Therefore, reduced breakdown of bradykinin in the tubes without gel results in lower concentrations of des-Arg9-bradykinin for these individuals compared to the concentrations obtained in tubes with gel separator. In agreement, des-Arg9-bradykinin concentrations after 24 h clotting (not shown) were higher in serum from tubes without gel for all individuals due to the absence of bradykinin and reduced proteolysis of des-Arg9-bradykinin in this tube type. The results for Hyp3-bradykinin showed the same pattern as for bradykinin, although lower concentrations were measured (not shown).
Stability in Serum
Similar as during the clotting process, continuing protease activities caused alterations in the serum concentrations when serum was left at RT after removal of the blood clot. The ITIH4-derived peptides were measured in serum from which the solid material was removed after clotting for 30 min and which was subsequently incubated at RT for different periods of time. All peptides showed similar patterns, concerning the changes in serum concentrations, compared to the effect of incubation at RT in the presence of the blood clot, as presented in Figs. 1 and 2. Moreover, for some individuals, identical concentrations were measured in serum left at RT for the same period of time either during or after clotting. Examples of the measured concentrations in serum from healthy individuals with similar incubation times at RT with or without the presence of the solid clot material are shown for ITIH4-21 and ITIH4-25 in Fig. 5.
Average relative changes in serum concentrations after incubation of serum from four healthy individuals for 4 h at RT
Relative difference (%) ±standard deviation
Clotting time (h); temperature (°C)
Tube with gel
Tube without gel
+ 48.3 ± 30
+ 7.4 ± 8
+18.9 ± 8
−54.5 ± 17
−55.5 ± 18
−64.3 ± 18
−53.9 ± 23
−82.9 ± 3
−87.1 ± 5
+ 151.6 ± 20
+ 298.1 ± 124
+ 95.3 ± 40
+ 75.4 ± 17
+ 417.5 ± 99
+ 361.7 ± 108
+ 35.2 ± 13
+ 30.0 ± 11
+ 202.5 ± 43
+139.0 ± 52
+ 90.4 ± 12
+ 127.7 ± 42
+ 109.4 ± 46
+ 71.9 ± 10
+ 33.1 ± 23
+ 18.8 ± 14
−10.9 ± 12
−11.7 ± 20
Intra- vs. Inter-Individual Variations
As partly discussed above and illustrated in Figs. 1, 2, 3, 4, and 5, large variations in the serum concentrations of the peptides were already observed between four healthy individuals. Lowest inter-individual relative standard deviations (RSD) in samples with the shortest clotting time were observed for Fib-α [605–629] and ITIH4-29 (10.8% and 14.1%, respectively), while variations were particularly high for bradykinin, des-Arg9-bradykinin, and ITIH4-28 (138, 57% and 44% RSD, respectively). Additionally, effects of clotting time on the serum concentrations of the various peptides were compared for one individual from whom blood was collected on three different days. Examples of intra-individual variations in C4a [1337–1350], des-Arg9-bradykinin, ITIH4-22, and ITIH4-27 serum concentrations are presented in Fig. 8.
These results suggest that prolonged incubation, allowing further proteolytic breakdown, likely increases inter-individual variabilities, while reducing intra-individual variations, although observed for only one individual at three different moments. Eventually, prolonged ex vivo processing possibly reduces the effect of confounding factors, providing a better reflection of one individual’s endo- and/or exoprotease activity as well as a better differentiation between protease activities in different individuals. However, it is difficult to say whether these variations are wanted (expressing variations in the activity of proteases of interest) or unwanted (caused by factors other than disease-related activities of proteases, e.g., coagulation status). Nonetheless, if variations between the four healthy individuals are only caused by haemostatic differences, one would expect similar inter-individual variations for all peptides. This is clearly not the case, e.g., the individual that showed much higher concentrations of ITIH4-22 after prolonged clotting (Fig. 2) than the other individuals, showed more or less similar protease dependent formation of the other ITIH4 fragments. This suggests interferences of more specific proteases than those solely depending on the coagulation and complement activation.
Moreover, the complicated history of the studied peptides is likely to contribute to the observed variations as their formation depends on (1) generation of founder peptides by ex vivo proteolysis, eventually activated during clotting, (2) cleavage of these founder peptides by exo- and or endoproteases, and (3) simultaneous breakdown of the peptide itself.
Comparison Between Breast Cancer Patients and Healthy Controls
Measured concentrations of the various peptides in serum from breast cancer (BC) patients and controls (CO) after direct measurement or after 24 h incubation at RT
After 24 h at RT
Median concentration [Range]a (ng/ml)
Median concentration [Range]a (ng/ml)
1.1 b [1–18]
0.5 b [1–5]
Owing to the recent development of two selective and specific LC-MS/MS methods for the absolute quantification of, in total, 13 proteolytic peptides in human serum, it was possible to accurately determine the specific influence of several sample handling variables on the serum concentrations of these putative cancer biomarkers. The results presented in this manuscript show that clotting time, temperature, and type of collection tube drastically affect the final serum concentrations. The different impact of these effects on the various peptides demonstrates the necessity of more specific sample preparation procedures to provide more consistent results and further investigate the large number of proteolytic peptides proposed as putative biomarkers for different types of cancer by various semi-quantitative studies. This study, therefore, very well provides suggestions for follow-up investigations to better explore the potential of these peptides as possible biomarkers.
Furthermore, similar ex vivo degradation and/or generation of the various peptides by proteases in serum as during coagulation showed that the protease activities are not solely depending on blood coagulation. This implies that other factors than haemostatic conditions are very likely to contribute to altered protease activities. Moreover, the increased discriminative value of the potential breast cancer marker ITIH4-22 after excessively long exposure to ex vivo proteolysis could support the hypothesis of tumor specific proteases and prolonged incubation of serum samples therefore deserves consideration for future quantitative comparisons of specific peptide fragments.
Ultimately, the presented study provides improved awareness of the pitfalls and opportunities of the low molecular weight peptidome in the search for novel cancer biomarkers.
We would like to thank all volunteers as well as the assistants at the UMC Utrecht, Saltro Diagnostic Center and SMC Papendal for donation and collection of the blood samples.
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