Inhibition of Intrinsic Proteolytic Activities ... - ACS Publications

Inhibition of Intrinsic Proteolytic Activities Moderates Preanalytical Variability and Instability of Human Plasma Jizu Yi,* Changki Kim, and Craig A. Gelfand BD Diagnostics, One Becton Drive, Franklin Lakes, New Jersey Received October 18, 2006

Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome. Keywords: bradykinin • C3 peptides • inhibition • mass spectrometry • proteolysis • stabilization • serum and plasma

1. Introduction Human plasma and serum are complex fluids with a wide dynamic range of proteins,1-2 including many enzymes, especially proteases.3 The enzymes can alter proteins within veins and contribute to the high heterogeneity of plasma proteins and peptides. Some of these proteins and peptides may be of value as biomarkers, with their presence/absence or relative abundances being correlated with health status and thus useful for prognosis or diagnosis. Recent proteomics-based studies of serum and plasma have revealed recognition patterns that can distinguish various types of cancers4-9 or other diseases10-11 from non-disease controls, suggesting new diagnostic methods for clinical applications. Human serum and plasma samples have been widely used in clinical diagnostics. While proteome-wide biomarker discovery continues to expand, some recent proteomics-based research has focused on the lower molecular mass proteins (LMMP) and peptides.12-15 Like large protein regimes, these LMMP and peptides may contain useful information for new diagnostics.16-18 However, arguments have been raised regarding whether or not the reproducibility, sensitivity, and specificity of SELDI-based recognitions pattern are sufficient for clinical * To whom correspondence should be addressed. Dr. Jizu Yi, BD Diagnostics, One Becton Drive, MC 305, Franklin Lakes, New Jersey 07417; Tel, 201-847-5462; Fax, 201-847-4851; E-mail, [email protected].

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application19, 20 since the first such serum peptide pattern for ovarian cancer was published.4 Intense criticism has also come from concerns over uncontrolled clinical and analytical variations.21-24 Increasing evidence indicates that plasma and serum proteins are subject to a wide variety of alternative and unstandardized preanalytical procedures and factors, including anticoagulants, centrifugation, sample processing, and storage conditions.25-29 Due to additional complications induced by the clotting process during serum preparation, EDTA and citrate plasma samples were recommended as the preferred samples for plasma proteome studies by researchers from the Human Proteome Organization (HUPO),14, 26 as a result of pilot studies from its Plasma Proteome Project (PPP). Sequence-based identifications of serum and plasma peptides suggested that many peptides were generated by proteolytic cleavage of plasma proteins to produce parental fragments by intrinsic endoproteases, then by multiple truncations of the parental fragments by aminopeptidases and carboxypeptidases.15, 16, 18 Although these peptides generated ex vivo in serum may contain diagnostic information,16, 18, 30 none of the available data on protein fragments as diagnostic markers is either strong enough or has passed validation until very recently.31 There remains the very important question of whether these peptide biomarkers are stable enough in traditional clinical settings for widespread diagnostic application. 10.1021/pr060550h CCC: $37.00

 2007 American Chemical Society

Inhibition of Intrinsic Proteolytic Activities

To date, instability of serum/plasma proteins and peptides due to intrinsic proteolysis during blood collection and subsequent sample preparation has not been fully or specifically evaluated. In this study, the ex vivo proteolytic degradation of plasma and serum samples was investigated by analyzing peptides, as an indicator of protein stability, using highresolution MALDI-TOF MS, and evaluating samples over a time-course incubation. High-resolution MS allows unambiguous tracking of individual peptides, facilitating comparisons between samples either from different collection tubes or over the incubation times. The relative peak heights or areas of the same peptide in the time-course experiment represent the relative abundance of the fragment as a function of time. Further, peptides can be identified by TOF/TOF analysis. We demonstrate that serum and plasma peptides can be dramatically affected by intrinsic proteolysis. Peptides are generated during the first minutes of sample collection and handling, suggesting rapid ex vivo proteolytic degradation of the blood proteins and peptides. Intrinsic proteases are responsible for both the peptide differences observed among serum and three anti-coagulated plasmas (with EDTA, citrate, or heparin) and the time-dependent changes of peptides after sample collection and processing in all samples. These proteolytic effects are suppressed by the inclusion of protease inhibitors (PIs) in an EDTA-plasma collection device. Our results demonstrate that immediate exposure of blood to PIs during blood collection helps preserve plasma proteins.

2. Methods and Procedures 2.1. Blood Collection and Plasma/Serum Preparation. Human blood from a healthy individuals was directly drawn into evacuated tubes including glass serum tubes without additives (BD Vacutainer, product number 366430), plasma tubes containing an anti-coagulant: either buffered sodium citrate (BD Vacutainer, product number 369714), lithium heparin (BD PST, product number 366643), K2EDTA (chemically similar to BD, product number 367525), or K2EDTA with a proprietary broad-spectrum cocktail of protease inhibitors formulated specifically for blood (BD P100, product number 366455/366456; for research use only, not for diagnostic use). The EDTA and P100 tubes also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. After the collection of the blood specimen, serum tubes were placed at room temperature to clot for 60 min and then centrifuged at 1500× g for 15 min at room temperature. Plasma tubes were spun immediately after blood was drawn (typically within 10 min), for 15 min at 1500× g, and at room temperature to minimize the handling condition and to prevent possible platelet activation. Both plasma and serum samples were pipetted out of the blood-collection tubes into Eppendorf microcentrifuge tubes and were either frozen within 15 min at -80 °C until use, or used immediately as specified. To minimize the dwell time during the sample process, the drawn blood is processed directly after acquisition from individual subjects in this study. Full acquisition and processing typically takes approximately 30 or 90 min for plasma and serum, respectively. Samples from 20 subjects were collected and processed over 4 days for this study. 2.2. Time-Course Incubation and Sample Preparation for MALDI-TOF Mass Spectrometric Analysis. The frozen serum and plasma were thawed in a room-temperature water bath for approximately 5 min. For “time 0” experiments, the thawed samples were immediately and biochemically quenched by

research articles addition of 1% TFA up to 0.1% final concentration. For timecourse experiments, 500 µL thawed samples were incubated at room temperature. A 45 µL aliquot was withdrawn at each specified time and quenched by adding 5 µL of 1% TFA solution. The quenched sample was subsequently transferred onto a Microcon YM-3 (Millipore) and spun in a Micro Centrifuge (Eppendorf Centrifuge 5417R) at 12500 rpm and 10 °C for 45 min. The filtrate was collected and desalted using Zip-Tip C18 (Millipore). The eluted peptides (1 µL) were mixed in 1:1 (v/v) ratio with 5 mg/mL of R-cyano-4-hydroxycynnamic acid (CHCA) as the matrix. The peptide mixture was spotted on a plate, air-dried, and analyzed using MALDI-TOF MS. 2.3. MALDI-TOF MS. The MALDI-TOF MS and MS/MS analyses were performed on an ultraflex II MALDI-TOF/TOF MS (Bruker-Daltonics). MS spectra of positive ions were recorded in an AutoXecut method and in reflector mode. The mass spectra were collected in a mass range from 800 to 3200 m/z with 25.1 kV in ion source 1, 22.1 kV in ion source 2, 9.6 kV in lens, 26.4 in reflector, and 13.9 kV in reflector 2. The digitizer and other instrument-specific settings were the same as the default settings of the instrument by the manufacturer. The final spectrum was calibrated against a spectrum obtained from seven calibrants on the well next to the sample. This external calibration method allowed us to reach a mass accuracy of better than 10 ppm. For relative or quantitative comparisons of spectra, the spectrum of each sample was obtained from accumulation of 30 qualified spectra, each of which was obtained from 100 laser shots. The sample site targeted by the laser was moved automatically after each of 100 shots to prevent over burning of the sample. During the accumulation, the quality of the spectrum from the 100 shots was evaluated in terms of peak resolution and signal-to-noise ratio. A spectrum with less than 1000 peak resolution was filtered out. Under these settings, reproducibility of the method from sample to spectrum was tested. Among 20 replicates of a P100 plasma sample, differences in peak intensities of individual peptides are less than 25% CV, and the differences are decreased to less than 7% CV after normalization using one peak as an internal standard. 2.4. MALDI TOF/TOF MS Analysis. Interesting peptides observed in MALDI-TOF MS, especially including the peaks either differing between sample types or changing over time, were selected for TOF/TOF analysis, to identify the peptide sequences. The most intense peak of a peptide in time-course MALDI-TOF MS was chosen for this analysis. MS/MS spectra were acquired using the default lift method provided by the manufacturer. Calibration of the method was performed every 6 months or whenever necessary. A TOF/TOF spectrum was further calibrated using the parent ion’s mass measured from TOF MS mode. During ion selection, the timed-ion-selector (TIS) window was set to a (10 Da width centered at the parent ion. 2.5. LC-ESI-MS/MS Analysis. The LC-MS/MS analysis was carried out on a Q-TOF micro (Waters) coupled with a reversephase chromatography system. A purified peptide sample was loaded on a reverse-phase C18 column with a size of 75 µm × 150 mm and eluted at a flow rate of 200 nL/min with a linear gradient of 3-95% acetonitrile (ACN) for 65 min. The Q-TOF MS was run using the automatic settings. MS survey was screened in 200-1990 (m/z) range, from which the 3 highest abundant ions, either its single- or a multiple-charge state of each ion, were selected for MS/MS analysis in 50-1990 (m/z) range. Journal of Proteome Research • Vol. 6, No. 5, 2007 1769

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Figure 1. MALDI-TOF mass spectra of peptides in human serum and plasma samples. Numbers above the peaks indicate the positions (m/z) of the monoisotopic peaks. Color coding indicates peak identifications: bradykinins (BK) in red, Fibrinogen (FPA or Fib) in green, C4- and C3-derived peptides (C4 and C3) in blue and black, respectively (also listed in Table 1). Gray labels indicate unidentified peptides.

2.6. Spectral Analyses and Database Search. The TOF or TOF/TOF spectra were processed by flexAnalysis (BrukerDaltonics) with median baseline subtraction and smoothing. The peaks were detected with SNAP algorithm and S/N threshold 3. The other parameters were the same as those in the default method. For comparison, all of the time-course spectra were processed using the same parameter settings. For a sequence analysis, the peak lists of MALDI-TOF/TOF spectrum were exported into BioTools (Bruker-Daltonics) and Q-TOF MS/MS profiles were into MassLynk (Water). The peptides of interest were identified by searching their monoisotopic ions in the NCBInr database using Mascot (Matrix Science, www.matrixscience.com) with the following settings: “Homo sapiens (human)” for taxonomy, “None” for enzyme, peptide charge of “1+”, peptide MS error tolerant of “( 0.4” Da, and MS/MS error tolerant of “( 0.8” Da. A matched sequence was accepted if the returned Mowse score was higher than 50.

3. Results and Discussion 3.1. Intrinsic Peptide Content: Serum and Plasmas. Human serum specimens are the most common samples used in clinical diagnostics. However, for proteomic and peptidomic studies, it has remained unclear whether serum or plasma is a more appropriate blood sample, particularly for biomarker discovery. Among the goals of the HUPO PPP effort was definition of the ideal sample and handling standard, with early findings that recommended plasma as the preferred sample.14, 26 1770

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In this study, we examined the peptide content of serum and plasma samples, focusing on the intrinsic peptide content of these samples. Our hypothesis is that the peptides and their time-course changes are a direct indication of proteasemediated degradation of their parent proteins or peptides, and thus, examination of these peptides is directly indicative of sample stability. By monitoring serum and plasma samples from “time 0” up to 3 days after collection, we tested whether peptides present in the drawn-blood samples were sufficiently stable for their eventual detection. For these studies, we collected blood samples from healthy subjects into a series of tubes from the same venipuncture event: a serum tube and plasma tubes with either heparin, EDTA, or citrate as the anticoagulant, with tube sequence randomized for each subject to eliminate any systematic errors. After processing as described in Methods and Procedures, the serum or plasma sample was quenched by adding TFA to 0.1% final concentration. The peptides were extracted using 3 kDa cutoff membrane filters and reverse-phase chromatography and were detected using MALDI-TOF MS. The peptide spectra of these four matched samples are shown in Figure 1, as one example of 10 subjects tested individually. Peptides observed in serum are dramatically different from those in the three plasma samples (Figure 1). A total of 137 peptide peaks were detected in the serum sample, approximately double the number observed in heparin (73 peaks), EDTA (66 peaks), and citrate (57 peaks) plasma samples. The peak intensities were also higher in the serum than in any of

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Inhibition of Intrinsic Proteolytic Activities Table 1. Identified Abundant Peptides and Their Parent Proteins in Human Blood Samplesa,b

a Listed peptides in serum, citrate, and heparin plasmas were observed in MALDI-TOF spectra of 10 healthy individuals, identified by either TOF/TOF MS or LC-Q-TOF MS/MS analysis. The presence of EDTA peptides is presented as the averaged peak intensity (MALDI-TOF MS) of 20 inidividual samples, and the presence of P100 peptides is presented as the ratio of the averaged peak intensity of the same 20 individuals from the P100 tubes over the EDTA tubes.b 9 stands for detectd or present in MALDI-TOF MS; 0 for not detected or absent; 9/0 for present in some individuals but not in others; “-” for ambiguous. c Observed monoisotopic MH+. d Serum peptides were also detected by Villanueva et al.19 e Heparin peptides were also analyzed by Koomen et al.16 f Peptide with the oxidation of the Proline at the third residue. g Not reported as the serum peptide in previous report;19 instead, the same mass peptide was reported with the “Arg” residue in the amino terminal end.

the plasmas. Highly abundant peaks in serum span from 1000 to 1600 m/z and include nine monoisotopic peaks at 1020.49, 1077.51, 1206.54, 1263.58, 1350.62, 1465.62, 1518.66, 1536.69, and 1616.64 m/z (Figure 1). Six of these peptides (green labels in Figure 1) have been matched to fibrinogen peptide A (FPA) (Table 1) and are sequentially one residue shorter at their amino-termini, consistent with previous reports.16,17,19 This laddering effect is consistent with the fact that the full-length peptide (at 1536.7 m/z) is generated as the expected ex vivo proteolytic product of clot formation and then truncated/ altered by nonspecific aminopeptidase activities.16,17,19 Further, this phenomenon of ex vivo protease and peptidase activities also represents a possible source of uncontrolled preanalytical variation that may be evident across other proteins and peptides. The full-length FPA (1536.7 m/z) and its one-amino-

acid-residue-shorter peptide (1465.7 m/z) were also observed in EDTA but only inconsistently in heparin or citrate plasma samples from all subjects (Figure 1). The other shorter FPAs were not detected in citrate and EDTA samples but were detected in some heparin samples (Table 1). Peaks observed in serum in a higher mass range spanning from 2500 to 3200 m/z, including 3190.33, 2931.21, 2768.16, and 2553.04 m/z (green labels in Figure 1), were matched to fibrinogen R chain (Table 1). These are sequentially one or two residues shorter at their carboxy-termini, suggesting that carboxypeptidases act on these peptides. Different peptide spectra were also observed among the three plasma samples (Figure 1). EDTA showed the fewest peptides in the higher mass range (2000-3000 Da) and the most peptides in lower mass range (1000-2000 Da), whereas Journal of Proteome Research • Vol. 6, No. 5, 2007 1771

research articles the heparin and citrate plasmas were similar in peptide content by peak positions. Both heparin and citrate samples have abundant peaks, more than the EDTA sample, at 2228.00, 2378.28, and 2659.26 m/z (Figure 1). The peak at 2659.26 m/z is mapped to the peptide generated from fibrinogen alpha chain (green labels in Figure 1, Table 1), likely due to a small amount of ex vivo coagulation; the peaks at 2378.28 m/z is mapped to complement C4 (blue labels in Figure 1), likely due to the activation of the complement pathway. The heparin sample also displayed more peaks than both citrate and EDTA plasma samples at mass range from 2000 to 2500 m/z, including 2011.03, 2115.10, 2184.47, 2228.06, and 2310.22 m/z, whose sequences were not determined in this study (gray labels in Figure 1). Compared to the heparin and citrate samples, the EDTA sample displayed more abundant peaks at 1098.577 and 2021.13 m/z, identified as complement C3 (black labels in Figure 1), and at 1896.05 m/z, matched to complement C4 (blue label in Figure 1). More peptides truncated from complement C3f peptide (2021.13 m/z) were also detected in the EDTA sample than in the other two plasmas (Table 1). Two other peaks, 1739.978 and 2378.24 m/z, with higher intensity in the heparin and citrate samples than in the EDTA sample, were also matched to complement C4 (Table 1). Because both C3 and C4 are key components of the complement pathway,32 these observations suggest that none of these three anticoagulants alone can fully inhibit the ex vivo activation of the complement pathway. The above results showed that intrinsic serum proteases generate peptides by ex vivo digestions of proteins during the clotting process and, accordingly, result in the substantial peptide difference between a serum and a plasma sample. Notable peptide differences were also observed among heparin, citrate, and EDTA plasma samples (Figure 1 and Table 1), which suggested a correlation between the anticoagulants and the plasma peptidome, or specifically between the anticoagulants and the intrinsic plasma protease activities. 3.2. Proteolytic Activity in Serum and Plasma. To further explore these intrinsic protease activities, we monitored the variability of peptide content as a function of time by incubating serum and plasma samples at room temperature. Figure 2A represents the peptide spectra (750-3020 m/z) of the serum sample after incubation for 0, 5, 24, and 72 h. Substantial changes in peak intensity and total peak number were observed over time. The intensities of the highly abundant peaks, especially the six FPAs observed at “time 0”, decreased over incubation time (Figure 2A), suggesting general instability of the serum peptides. Interestingly, the intensity of some peaks increased during early incubation time and then decreased later on. This time-dependent change of concentration represents the characteristic of an intermediate of a sequential reaction. Two such peaks, 2931.21 and 2768.155 m/z (Figure 2A), are matched to SSSYSKQFTSSTSYNRGDSTFESKSY and SSSYSKQFTSSTSYNRGDSTFESKS, respectively (Table 1). The first peptide is generated from its parental peptide or protein, and then its C-terminal residue (Y) is removed by a carboxypeptidase producing the second peptide. This second peptide is further truncated on its C-terminal end into the smaller fragment, SSSYSKQFTSSTSYNRGDSTFES (2553.043 m/z) (Figure 2A and Table 1), which is further turned to smaller peptides. Therefore, the time-course MS results are consistent with mechanistically sequential truncations of peptides by carboxypeptidases. Furthermore, a magnified view of Figure 2A 1772


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indicates that the intensities of initially lower abundance peaks at “time 0” increase with time, and new peaks are also generated (Figure 2B), suggesting that the serum proteins and/ or larger peptides are proteolytically digested in a continuous and ongoing manner during incubation. To confirm that these observations were universal, we repeated the same experiment with serum samples collected from 10 subjects representing a range of dietary states (pre- and postprandial) and found similar results in all 10 samples tested individually (data not shown). The results clearly indicate a general instability of serum proteins and peptides in clinical settings. Similar experiments were performed with EDTA, citrate, and heparin plasma samples, collected at the same time as the serum samples from the same 10 subjects. A representative time-course comparison (Figure 3A-C, and supporting Figures 3A-S, 3B-S, 3C-S, Supporting Information) shows that peptide changes over time are always observed in all three plasma samples, although these changes are not as extensive as in the serum samples (compare Figure 3A-C with Figure 2B). Some of the peptides were detected at only one specific time point (f, Figures 3B,C) and some others first increased and later decreased in intensity (g, Figures 3A-C), both representing the intermediates of sequential reactions as described above for serum (Figure 2A). Some of the peptides observed at “time 0” disappeared at 5 h or decreased over time (open arrows, Figure 3A-C), indicating that these were substrates for subsequent proteolytic digestion. Some peptides were newly generated over time, and/or their intensities increased over time (filled arrows, Figure 3A-C), representing proteolytic products. All of these observations are consistent with ongoing and sequential proteolysis in all of the plasma samples, with peptides being simultaneously cleaved first from their parent proteins or larger peptides and these peptides being subsequently degraded through multiple truncations by other proteolytic enzymes, specifically by peptidases. As a consequence, few peptides are stable over incubation time. Comparing the three plasma samples, we can see that specific anticoagulants are associated with certain unique peptide changes over time. In the mass range of 2000-3000 m/z, the citrate and heparin samples display both more peaks and increasing peak intensities over time than the EDTA sample (supporting Figure 3, Supporting Information), including the peptides cleaved from fibrinogen alpha (2659.29 m/z) and complement C4 (2378.21 m/z) (Table 1 and supporting figures). The results indicate that EDTA appears to be a better inhibitor to prevent the generation of the peptides in this mass window (2000-3000 m/z). However, in the lower mass range of 9002000 m/z, the EDTA sample displays the most peaks among the three plasmas, and the extra peptides have been matched to complement C3 and FPAs (Table 1). Nevertheless, the peaks observed in the EDTA sample still exhibit similar, if not more, time-course changes as those observed in the citrate and heparin samples (comparing Figure 3A-C, and supporting figures). The above results show that the peptide variability is muted in plasma versus serum, supporting previous recommendations that plasma is the preferred sample.14,26 However, intrinsic plasma protease and peptidase activities result in easily detectable preanalytical variability and instability of the plasma samples as well, with each anticoagulant having a unique “signature”. Although each of the three anticoagulants is indeed a protease inhibitor, in particular of the clotting cascade enzymes, none fully inhibits intrinsic plasma proteolysis. Con-


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Figure 2. Time-dependent proteolytic digestion of serum peptides. (A) Peptide spectra were obtained after incubating the serum samples for specified time periods. (B) Magnified view of the highlighted area in (A). Note that “0 h” refers to a rapidly prepared serum sample, which actually exists ex vivo for approximately 1 h and 20 min, accounting for serum preparation, including incubation for clotting at room temperature for 60 min and centrifugation for 15 min. The abundant peaks observed at “ 0 h” decrease over incubation time whereas some peaks increase or are generated with time (A and B), and others, such as 2768.155 and 2931.21 m/z, increase first and decrease later on (A). Journal of Proteome Research • Vol. 6, No. 5, 2007 1773

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Figure 3. Time-dependent proteolytic digestion of plasma peptides. Plasmas from the same subject, including EDTA (A), citrate (B), and heparin (C) as anticoagulants, were incubated for the time specified. Peaks are observed to be decreasing or disappearing (open arrow), or increasing (filled arrow), or increasing then decreasing (g) with incubation time. Some peaks are observed at only a specific time point (f). The spectra are magnified to highlight the extent of changes among both higher and lower abundant peptides. Full spectra are included as Supporting Figures 3A-S, 3B-S, and 3C-S where the highlighted areas are respected to Figures 3A, 3B, and 3C. One experimental representative of 10 samples tested individually is shown. 1774



sidering that it takes at least 15 min to collect blood and process it into plasma by centrifugation, we expect that rapid proteolytic activities intrinsic to plasma samples can cause significant degradation. One illustration of this very rapid proteolysis is the fact that the differences of peptide spectra among three plasmas are evident at our “time 0” (Figure 1), representing approximately 15-20 min during sampling. Thus, even the most time-efficient blood collection and sample processing cannot overcome plasma variability caused by intrinsic proteolytic enzymes. 3.3. Stabilization of Plasma Proteins by Protease Inhibitors. Sequence analysis suggests that much of the peptide content of the plasma/serum proteome results from cleavage of parental proteins by specific endoproteases (e.g., thrombin and plasmin), consistent with previous observations,15, 18 but the specificity and/or variability of such processes remains unclear. Further, our current data shows that both intrinsic and ex vivo generated peptides can be digested by aminopeptidases and/ or carboxypeptidases. Aminopeptidase activity seems to dominate, as more daughter peptides (e.g., FPA and C3-derived peptides) are generated from the removal of N-terminal residues than C-terminal residues (Table 1). The widespread nature of peptidase-caused damage suggests that these peptidase activities are likely nonspecific toward their peptide substrates. To better preserve plasma proteins, we have included a cocktail of protease and peptidase inhibitors (PIs) in a plasmacollection tube (BD P100) containing EDTA as the anticoagulant. An important aspect of this tube is that the blood is mixed with the PIs immediately upon blood collection, allowing the PIs to immediately inhibit proteolytic degradation. We examined the effects of PIs on the plasma peptides by directly comparing the P100 and EDTA samples from the same 10 subjects, both at “time 0” and over the same incubations. 3.3.1. Benefits of Protease Inhibitors at “Time 0”. Plots of averaged spectra from the 10 individually tested P100 and EDTA samples highlight several notable differences between the two samples at “time 0” (Figure 4A). Fewer peaks and lower peak intensities in the mass range from 840 to 1200 m/z were observed in the P100 than in the EDTA plasma, as shown in a typical magnified plot of the two samples from the same single subject (Figure 4B), whereas higher peak intensities from 1500 to 1880 m/z were observed in the P100 samples (Figure 4A). Several peptides within these spectra are of particular interest, including bradykinin (labels in red), fibrinogen peptide A (green), and peptides derived from complement C4 (blue) and complement C3 (black). 3.3.1.1. Bradykinin. Plasma bradykinin, BK[1-9], cleaved from high molecular weight kininogen (HMWK) by kallikrein, is a vasoactive peptide involved in cardiorenal physiology and inflammatory states and is linked to the pathophysiology of hypertension and diabetes.35 The determination of BK levels correlated with disease states has been hampered by its artificially high concentration in drawn blood due to the activation of the kallikrein-kinin system induced by vascular trauma and negatively charged surface (e.g., glass) of blood collection containers.33, 34 Further complicating the issue, BK[1-9] has also a short-lived nature due to fast metabolic degradation by multiple peptidases.34 Two peaks at 1060.579 and 1076.578 m/z were identified (via MS/MS, data not shown) as full-length bradykinin (BK[1-9]) and its oxidized state (O-BK[1-9]), respectively (Figure 4B and Table 1). The two peptides were easily detected with relatively

research articles high abundance in the EDTA samples, whereas in the P100 samples, both BK[1-9] and O-BK[1-9] were either not detectable (Figure 4A,B) or detected with considerably reduced abundance (8.5-15% of intensity in EDTA), based on averaged results of 20 samples (Table 1). This evaluation confirms that the high levels of the BK peptides in the EDTA sample are artificially generated during and after blood collection and plasma preparation. Furthermore, peaks of BK[1-8] at 904.46 m/z and its oxidized analog O-BK[1-8] at 920.46 m/z, most likely truncated from BK[1-9] and O-BK[1-9] by carboxypeptidases N and M,35 are not visible in P100 but are detected in the EDTA plasma (Figure 4A,B) as well as in the citrate, heparin, and serum samples (Figure 1 and Table 1). These observations indicate that both the ex vivo proteolytic generation of BK[1-9] and O-BK[1-9] by plasma kallikreins and ex vivo proteolytic degradation of these peptides by carboxypeptidases are effectively inhibited in P100. Although the artifacts of typical blood sampling mask a potential utility of BK, our results indicate that immediate exposure of the blood sample to protease inhibitors may enable the use of intrinsic plasma BK as a pharmaceutical and clinical biomarker. 3.3.1.2. Fibrinogen Peptide A. Interestingly, the full-length fibrinogen peptide A (fl-FPA, ADSGEGDFLAEGGGVR, 1536.690 m/z) and its shortened peptide with the N-terminal Ala removed likely by an aminopeptidase (s-FPA, DSGEGDFLAEGGGVR,1465.623 m/z), usually observed in a serum sample (Figure 1 and Table 1), were unexpectedly observed in both EDTA and P100 samples (Figure 4A and Table 1) although at greatly reduced levels compared to serum (Figures 1 and 2A). We hypothesize that the mechanical separator (standard feature of P100 tubes and also used in the EDTA tubes as a control for this study), which functions in isolating cells from plasma, leads to the generation of this small amount of FPA, because FPA is not observed in a standard EDTA sample without the mechanical separator (data not shown). fl-FPA has a slightly higher peak intensity in the P100 than in the EDTA sample (P100/EDTA ) 1.50), whereas s-FPA has a lower intensity in the P100 plasma (P100/EDTA ) 0.54) (Figure 4A and Table 1), suggesting that the PI cocktail in P100 tube displays the inhibitory effect on the aminopeptidase(s) responsible for truncating fl-FPA. 3.3.1.3. Complement C4. Among the three peptides identified as being from complement C4 (Table 1), two of these exhibited a reduced abundance in P100 compared to EDTA samples. The first C4 peptide, at 1896.01 m/z, observed in both EDTA and P100 (Figure 4A, also in serum, citrate and heparin samples (Table 1)), was confirmed by LC-MS/MS to be NGFKSHALQLNNRQIR (Figure 5), consistent with previous reports from heparin plasma.15 However, the result differed from what was observed in sera where the same peak was matched to RNGFKSHALQLNNRQI with the Arg residue at the N-terminal end.18 The second peptide identified with the truncated Arg residue (NGFKSHALQLNNRQI, 1739.95 m/z) was also detected in both EDTA and P100 samples. The intensity of the parental peptide is reduced in P100 by 8% (Figure 4A and Table 1), implying some inhibition by PIs of C4 activation. 3.3.1.4. Complement C3. Ten peptides of complement component C3 were identified in both the P100 and EDTA samples, representing the parental peptide (SSKITHRIHWESASLLR, C3f, at 2021.128 m/z) and a series of peptides sequentially shortened from either or both the termini of C3f (Figure 4A and Table 1). Among them, six peptides were observed with enhanced abundances in P100, including the full-length parental peptide C3f and the four largest daughter Journal of Proteome Research • Vol. 6, No. 5, 2007 1775

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Figure 4. Effects of protease inhibitors on the plasma peptides at “time 0”. The monoisotopic peak mass and sequence are indicated, with known identifications indicated by text color (bradykinins in red, fibrinogens in green, C4 peptides in blue, and C3 peptides in black). (A) Peptide spectra are overlaid from averaged spectra of 10 P100 samples (red) and 10 EDTA samples (blue) from same 10 subjects. Notable differences are observed between the P100 and EDTA samples across the entire peptide mass range. (B) Typical spectra of P100 and EDTA samples from the same subject are magnified in the mass range of 1025-1300 m/z. Bradykinin peaks, including 1060.579, 1076.578, 920.467, and 904.469 m/z, are easily detectable in the EDTA sample, whereas the same peaks are not detectable in P100 samples. 1776



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Figure 5. ESI-MS/MS profile of the peptide at 1896.01 m/z observed in both EDTA and P100 samples. The sequence is identified to be C4 peptide (Mowse score of 63), and its matched y and b ions are indicated.

peptides (SKITHRIHWESASLLR, ITHRIHWESASLLR, SSKITHRIHWESASLL, and SKITHRIHWESASSLL) with the ratios of peak intensity (P100/EDTA) of 9.77, 3.20, 4.33, 10.02, and 4.88, respectively (Table 1). Meanwhile, the three shortest peptides (HWESASLLR, WESASLLR, and HWESASLL) are lower in the P100 sample (Figure 4A and 4B) with intensity ratios (P100/ EDTA) of 0.12, 0.00, and 0.047, respectively (Table 1). These results provide another example of the inhibition of intrinsic plasma aminopeptidases by the inhibitors in P100. Interestingly, five C3 peptides (ITHRIHWESASLLR, THRIHWESASLLR, IHWESASLLR, HWESASLLR, and WESASLLR), which are generated from sequential truncations at the N-terminal end of C3f and themselves are subject to further truncations, are not consistently detectable in serum, citrate, and heparin samples (Table 1). This observation can by explained by four possibilities: (i) the five intermediate peptides are not stable due to further rapid truncation or degradation; (ii) the ex vivo generation of these peptides from their parental peptides is inhibited; (iii) the ex vivo generation of the original parent peptide (C3f) is inhibited; and (iv) the generation of C3f is not inhibited but this parent and all five daughter peptides are digested extremely quickly. In the sera used in this study, no chemical additives were present so that there was no inhibitory effect on the peptide changes. We also observed that peptides are subject to more extensive digestions in a serum than in a plasma sample (Figures 2 and 3). Furthermore, six highabundance FPAs in the serum sample decay quickly in the time-course experiment (Figure 2B), supporting the instability

of serum peptides. Therefore, we conclude that the absence of these five C3-derived peptides in serum is due to rapid degradation by intrinsic serum peptidases. In the citrate and heparin plasma samples, however, the original parent peptide C3f of these intermediate peptides was not consistently detected (Table 1). Therefore, either the ex vivo generation of C3f is inhibited most likely by the citrate and heparin used for anticoagulation in both samples (the third possibility), or extremely quick peptidase digestion has consumed C3f as well as the five intermediates (the fourth possibility).18 In both EDTA and P100 samples, C3f is always observed (Table 1 and Figure 4A), demonstrating that neither EDTA as an anticoagulant in these two tubes nor the protease inhibitor cocktail in P100 tube inhibits the ex vivo generation of C3f. The enhanced peaks of C3f and its longer derived peptides observed in the P100 sample indicate that the PIs slow down the fast degradation of these peptides and, thus, provide a longer life of these intermediates. As listed in Table 1, C3-derived peptides are observed primarily in the protease-inhibited plasma sample (P100), whereas FPAs are observed primarily in the PI-free serum sample (Table 1). The intensity patterns in these highly abundant FPAs and C3 peptide families in serum were found to have value as biomarkers to differentiate cancer patients from healthy controls.16, 18 Our observations of peptides as a function of time (sample aging) indicate the instability of the peptides in normal serum samples (Figure 2), and, by contrast, the largely increased stability in protease-inhibited plasma (comparing Figure 2 to Figure 6). Given the instability we see Journal of Proteome Research • Vol. 6, No. 5, 2007 1777

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Figure 6. Time-course peptide spectra of EDTA (A) and P100 (B) plasma samples. Representative time-course data is shown from one subject of the 10 examined in this study 1778


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in unprotected serum, it is important to test if these potential serum-based cancer biomarkers are sufficiently stable for routine clinical application. Also, with the C3-derived peptides stabilized in the inhibited plasma, it is worthwhile to test if the same or similar C3-derived peptides as cancer biomarkers discovered in unprotected serum samples are maintained in the protease-inhibited serum or plasma sample. If so, the improved stability of these biomarkers may provide a better opportunity for validation and clinical utility. Furthermore, because at least two C3 peptides (SKITHRIHWESASSLL and HWESASLL) are consistently detected in sera, three commonly used plasmas, and P100 plasma (Table 1), some of these 10 C3 peptides may represent the native peptides circulating in blood (in vivo). The generic preservation of the peptide content is evidenced by the considerably reduced smaller peptides (840-1200 m/z) but increased larger peptides (1025-1300 m/z) in P100 samples compared with those in EDTA samples (Figure 4A and Table 1). As abundant peptides are mainly ex vivo generated and may mask the diagnostic information from intrinsic peptides in lower abundance, it is rational to expect that both prevention of ex vivo peptide generation and preservation of the actual in vivo peptide content may lead to better biomarker discovery and eventually new diagnostic assays. Importantly, all of the above differences, including bradykinins, FPAs, C4- and C3-derived peptides, were observed from the “time 0” state of the samples. These extensive and obvious differences represent the effects of the PIs during the first ex vivo minutes of the sampling, indicating the benefits of immediate mixing of blood with the PIs. Although the benefits were observed on peptides derived from high abundant proteins, similar effects of PIs are expected on lower abundance proteins and peptides as well. Despite the stability provided by the broad spectrum of the protease inhibitors, a limited amount of residual protease and peptidase activities remain in the inhibited plasma (Figure 6B). Knowing the complexity of the plasma proteome, it is perhaps not surprising that achieving complete inhibition is a challenge, especially while retaining the typical requirements (e.g., low hemolysis level) for drawn blood samples and also without forming PI-protein adducts, which may shift protein spots as observed in previous 2D gel studies.26 For the latter topic, our studies to date with intact proteins (unpublished results) do not show any such adducts. 3.3.2. Effect of Protease Inhibitors during Sample Handling. We studied the stability of the protease-inhibited plasma using similar time-course incubation experiments as described above. The MALDI-TOF peptide profiles of the two fresh EDTA and P100 samples from one of our 10 subjects, processed immediately after centrifugation, are shown in Figure 6A,B. At this “time 0”, we observe generally lower peak intensities and fewer peaks spanning from 900 to 2100 Da in P100 than EDTA samples, and few detectable peptides from 2100 to 3200 Da in both samples (not shown). At 30 min, similar spectra were observed, but with a consistent observation of lower peak intensities in P100 than in EDTA. The P100 spectrum at “time 2 h” is comparable with the EDTA spectrum at “time 0”. After 2 h, several new peaks appeared (e.g., 1051.74, 1334.94, and 1404.94 m/z), which increase in intensity at 4 h but maintain a lower peak intensity in P100 than EDTA samples (Figure 6). Specifically, the peak at 1060.61 m/z, identified as bradykinin, BK[1-9], was first detected at 4 h in P100 (and still with lower intensity than the EDTA sample at “time 0”) and increased

thereafter, confirming that the ex vivo generation of this peptide is effectively inhibited for at least 2 h. The peptides identified as C4 (1896.07 m/z) and C3f (2021.10 m/z) also increase at 4 h. Still more peaks appeared after incubation for 24 h up to 48 h in both P100 and EDTA samples, whereas other peaks first increased then eventually decreased (e.g., 1448.98 m/z). Few peaks seemed to be stable for such long incubations. However, the P100 spectrum at “24 h” is comparable with EDTA spectrum at “4 h”. Overall, although the peak intensities and peak numbers still change in P100 sample due to residual proteolysis, these changes are much slower than in EDTA sample, demonstrating that the protease inhibitors provide a benefit both by reducing variability in the protein and peptide content and by minimizing time-dependent ex vivo changes. Specifically, the observation that there are no significant peak changes in P100 sample from 0 to 2 h suggests that BD P100 plasma peptides are reasonably stable for at least 2 h at room temperature, whereas the EDTA sample is measurably destabilized by our first incubation time point of 30 min. On the basis of these observations, we recommend the use of a protease-inhibited plasma tube, such as BD P100, for plasma proteomics studies, followed by sample analysis within a 2-hour time frame. If analysis cannot be performed within this time frame, the sample should be frozen and stored at -80 °C as a common practice.26, 29 Furthermore, if it is not feasible to use or freeze the samples within the 2-hour window, the P100 protease inhibitor cocktail helps to limit sample variability, by comparison to the three commonly used plasmas, for as much as 24 h at room temperature (Figures 3A-C and 6A,B).

4. Conclusions Our results from both the time-course MS analysis and sequencing analysis of the serum and plasma peptides are consistent with the fact that the peptides first generated due to ex vivo activation of the clotting cascade, including the kallikrein-kinin system and complement pathway, are subsequently altered by aminopeptidases and carboxypeptidases. These intrinsic protease and peptidase actions occur during the first minutes of blood collection and sample preparation, accounting for the peptide differences observed at “time 0” among the serum and three (EDTA, citrate, and heparin) anticoagulated plasma samples (Figure 1), despite the fact that the anticoagulants provide a partial inhibitory effect. The timecourse experiments confirm intrinsic proteolytic degradation in both serum and plasma samples, being more extensive and intensive in serum than in the plasma samples (Figures 2 and 3). Residual plasma protease and peptidase activities still result in time-dependent variations and instability of the samples including EDTA plasma. During blood collection and serum/ plasma preparation, the peptides are being generated by intrinsic endoproteases from their parental proteins, and simultaneously, both the ex vivo generated and intrinsic peptides are being truncated sequentially by peptidases from their either or both N-terminal and C-terminal ends. Each of these peptides is an intermediate of a sequential reaction, and as a consequence, few peptides are stable over time (Figures 2, 3, and 6). Importantly, the protease inhibitors included in P100 modulate and suppress these intrinsic protease and peptidase activities, providing a better native-like plasma at “time 0” (Figure 4). The inhibitory effect on peptidases is evidenced by the observations that the protease inhibitors enhance the intensities of larger peptides while reducing their shortened Journal of Proteome Research • Vol. 6, No. 5, 2007 1779

research articles peptides (Figure 4A,B). The protected peptides include fl-FPA and complement C3-derived peptides (Table 1). Meanwhile, the inhibitory effect on intrinsic endoprotease activities is evidenced by the observation that the P100 sample suppress the generation of the initial peptides from their proteins, including BK[1-9] and complement C4 peptides (Table 1, Figure 6A,B). At room temperature, the inhibitors stabilize the plasma for at least 2 h, providing a reasonably safe window of time for blood sample processing. Beyond 2 h, e.g. at 24 h, variations are still observed but are greatly reduced in the inhibited plasma compared with unprotected serum or plasmas (Figures 2, 3, and 6). Complement C3-derived peptides were recently described as potential cancer biomarkers in serum samples.16, 18 Interestingly, the most enhanced C3-derived peptide content is observed in the protease-inhibited plasma (Table 1), indicating that P100 stabilizes C3-derived peptides for potential clinical use. Significantly, stabilization of bradykinin, which is involved in cardiorenal physiology, inflammatory states, hypertension, and diabetes,35 is achieved through the inclusion of the protease inhibitors in P100. A greatly reduced or undetectable level of this peptide in the protease-inhibited plasma sample (compared to much higher levels in all other samples) indicates that the kallikrein-kinin system is inhibited effectively by the PIs in P100 tube (Table 1, Figures 4A,B and 6B). Therefore, P100 provides a means both to preserve the intrinsic BK[1-9] for a potential clinical use and to prevent ex vivo digestion of high molecular weight kininogen by kallikreins, which generate artefactual BK after sample acquisition. These specific examples, along with generic stabilization of peptides, indicate that the protease-inhibited plasma should provide a more robust protein and peptide sample for biomarker discovery and clinical analysis.

Acknowledgment. We thank Thomas Finocchio for critical assistance in sample collection, Diana Queiros and Doris Winter in graphic preparation, and the entire BD Proteomic team for support and critical review. Supporting Information Available: Figures 3A-S, 3BS, and 3C-S. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Anderson, N. L.; Anderson, N. G. The Human Plasma Proteomes History, Character, and Diagnostic Prospects. Mol. Cell. Proteomics 2002, 1, 845-867. (2) Omenn, G. S.; States, D. J.; Adamski, M.; Blackwell, T. W.; Menon, R.; Hermjakob. H.; et al. Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database. Proteomics 2005, 5, 3226-3245. (3) Walsh, P. N.; Ahmad, S. S. Proteases in blood clotting. Essays Biochem. 2002, 38, 95-111. (4) Petricoin, E. F.; Ardekani, A. M.; Hilt, B. A.; Levin, P. J.; Fusaro, V. A.; Steinberg, S. M.; Mills, G. B.; Simone, C.; Fishman, D. A.; Kohn, E. C.; Lotta, L. A. Use of proteomic patterns in serum to identify ovarian cancer. Lancet 2002, 359, 572-577. (5) Adam, B. L.; Qu, Y.; Davis, J. W.; Ward, M. D.; Clements, M. A.; Cazares, L. H.; Semmes, O. J.; Schellhammer, P. F.; Yasui, Y.; Feng, Z.; Wright, G. L., Jr. Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men. Cancer Res. 2002, 62, 3609-3614. (6) Ebert, M. P. A.; Ebert, M. P.; Meuer, J.; Wiemer, J. C.; Schulz, H. U.; Reymond, M. A.; Traugott, U.; Malfertheiner, P.; Rocken, C. Identification of gastric cancer patients by serum protein profiling. J. Proteome Res. 2004, 3, 1261-1266.

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