Prolyl 4-Hydroxylation of α-Fibrinogen

Abstract

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL),” is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of α-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated α-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated α-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated α-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.

For comprehensive analysis of plasma proteins, it is necessary to compare a sufficient number of blood samples to avoid simple interindividual heterogeneity, because the protein content of human samples varies significantly among individuals. Also, the provision of sufficient power is needed to detect protein modification because many plasma proteins undergo changes in the bloodstream (1). Even though the proteomic technologies have advanced (2, 3), there remains room for improvement. Different isotope labeling and identification-based methods have been developed for quantitative proteomics technologies (4–6), but the number of samples that can be compared by the current isotope-labeling methods is limited, and identification-based proteomics is unable to capture information regarding unknown modifications.

A label-free proteomics platform developed in our laboratory, termed “Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)² (7), simply compares the liquid chromatography and mass spectrometry (LC-MS) data and detects a protein modification by finding changes in the mass to charge ratio (m/z) and retention time (RT). Enhanced methods for accurate MS peak alignment across multiple LC runs have enabled the successful implementation of clinical studies requiring comparison of a large number of samples (8, 9). Using 2DICAL to analyze plasma samples of pancreatic cancer patients and healthy controls, novel prolyl hydroxylation of α-fibrinogen was successfully discovered.

Fibrinogen and its modification has been investigated because of its clinical importance (10, 11). On the other hand, prolyl hydroxylation has attracted attention after the discovery of the hypoxia-inducible factor 1α (HIF1α) prolyl-hydroxylase and its role in switching of HIF1α functions (12). Prolyl hydroxylation in other proteins has been energetically sought, but only a few such proteins have been identified (13). Only one study has reported prolyl hydroxylation of fibrinogen at the β chain (14).

Here, we report the detection of prolyl 4-hydroxylated α-fibrinogen by plasma proteome analysis, a protein modification that dynamically changes in plasma depending on the clinical status and is a candidate plasma biomarker.

EXPERIMENTAL PROCEDURES

Clinical Samples

Seventy-seven plasma samples (38 patients with pancreatic ductal adenocarcinoma and 39 healthy controls) were obtained from the National Cancer Center Hospital (Tokyo, Japan) (Sets 1 and 2), and 9 plasma samples (5 patients with pancreatic ductal adenocarcinoma and 4 healthy controls) were obtained from the Tokyo Medical University Hospital (Tokyo, Japan) (Set 3) (15). 685 plasma samples from patients with various diseases and healthy controls (Sets 4) were collected prospectively from seven medical institutions associated with the “Third-Term Comprehensive Control Research for Cancer” and will be described in detail elsewhere.³ Written informed consent was obtained from every subject. The study was reviewed and approved by the ethics committee of each institute.

Sample Preparation

To 20 μl of a plasma sample, 900 μl of phosphate-buffered saline and 100 μl of Con A-agarose (Vector, Burlingame, CA) were added, and the sample was incubated at 4 °C for 2 h. After extensive washing with phosphate-buffered saline, proteins bound to Con A were eluted by competition with 100 mm mannose. To 30 μl of the eluted sample, 10 μl of 5 m urea, 2.5 μl of 1 m NH₄HCO₃, and 3.3 μg of sequencing grade modified trypsin (Promega, Madison, WI) were added. After digestion at 37 °C for 20 h, peptides were dried with a SpeedVac concentrator (Thermo Electron, Holbrook, NY) and then dissolved in 50 μl of 0.1% formic acid.

LC-MS

LC-MS and data acquisition were performed as reported previously (7, 8). Briefly, the peptide samples were separated with a linear gradient from 0 to 80% acetonitrile in 0.1% formic acid at a flow rate of 200 nl/min for 60 min using the splitless nano-flow HPLC systems (DiNa (KYA, Tokyo, Japan)) (16). MS spectra were acquired every second in triplicate with nano-electrospray (nanoESI)-QTOF-MS (QTOF Ultima (Waters, Milford, MA)).

Peak Alignment Across Multiple LC-MS

MS peaks were detected, normalized, and quantified using the in-house 2DICAL software package, as described previously (7). To increase the accuracy of peak alignment across multiple LC-MS runs, we applied a greedy algorithm, which had been used for fast DNA sequence alignment, to supplement our previous method (8, 9).

Protein and Modification Identification

MS and MS/MS data were acquired by preparative LC-MS runs with a tolerance of ±0.1 m/z and ± 0.5 min of RT using QTOF Ultima and linear ion trap (LTQ)-Orbitrap (Thermo Fisher Scientific, Waltham, MA) mass spectrometers. The MS/MS data were analyzed with Mascot software (Matrix Sciences, London, UK) including oxidized histidine, oxidized methionine, and hydroxyproline as possible modifications. Chemical formulas were determined with Xcalibur software (Thermo Fisher Scientific) with mass tolerance of 5 ppm.

Cell Lines

Primary cultured normal hepatic cells (hNHeps) were purchased from Takara Bio (Shiga, Japan). KIM-1 was kindly provided by Dr. Masamichi Kojiro (Kurume University, Kurume, Japan). Hep3B was obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). HLE was obtained from the Health Science Research Resources Bank (Osaka, Japan). SK-Hep-1, Jhh-7, Hep-G2, HuH-7, and HuH-6clone5 were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

RNA Interference

Three siRNAs targeting each of the P4HA1, P4HA2, P4HA3, P4HB, EGNL1, EGNL2, and EGNL3 genes, as well as 2 control RNAs, were designed by Applied Biosystems (Foster City, CA). Cells were transfected with the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) (17). Knockdown of relevant mRNA expression was confirmed by real-time PCR at 24 h after transfection (16).

Antibodies

Anti-fibrinogen antibody (A0080) was purchased from DAKO (Glostrup, Denmark). GANP transgenic mice (18) were immunized with a synthetic peptide ESSSHHP(O)GIAEFPSR (P(O), hydroxyproline) (named HyP-ESS) conjugated to keyhole limpet hemocyanin. Monoclonal antibodies were generated by a standard cell fusion technique. The reactivity and titer of antibodies to HyP-ESS as well as unmodified (ESS) peptides were assessed by an antibody capture assay (19) using OPD (orthophenylenediamine) as a substrate (supplemental Fig. S6A).

Immunoblotting

Protein samples were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Blots were visualized with an enhanced chemiluminescence kit (GE Healthcare, Bucks, UK) and quantified as described previously (20).

Competitive ELISA

100 μl of plasma diluted 20-fold with phosphate-buffered saline or 100 μl of serially diluted HyP-ESS standard peptide were incubated with 100 μl of 1 μg/ml horseradish peroxidase-conjugated 11A5 antibody for 30 min. 50 μl of the solution was added to 96-well microtiter plates precoated with 50 ng of HyP-ESS peptide and incubated for 1 h. After extensive washing, wells were incubating with the OPD solution for 10 min, and color absorbance at 490 nm was measured (supplemental Fig. S6D).

Statistical Analyses

Mann-Whitney U test was performed with the open-source statistical language R (version 2.7.0) (9).

RESULTS

Large Scale Quantitative Plasma Proteomics of Pancreatic Cancer Patients

77 plasma samples (39 from patients with pancreatic cancer and 38 from healthy controls) were obtained from National Cancer Center Hospital. We used concanavalin A (Con A) to concentrate plasma glycoproteins (21). This “glycocapturing” procedure removed albumin and reduced the concentration of other abundant plasma proteins (22). Various aberrations of protein glycosylation accumulate in cancer (23, 24). Most tumor markers of pancreatic cancer used clinically, including CA19-9, DUPAN-2, and NCC-ST-439, are known to be carbohydrate antigens (23, 25). Each sample was anonymized, randomized, and measured in triplicate by 2DICAL. A total of 115,325 independent MS peaks were detected within mass ranges of 250–1600 m/z and an LC RT of 0–45 min (Fig. 1A). The correlation coefficient (CC) and coefficient of variance (CV) values for the triplicate data were over 0.95 and under 0.15, respectively, in most subjects. To increase statistical robustness, 77 samples were separated at random into two experimental sets (Set 1 (18 pancreatic cancer patients and 19 healthy controls) and Set 2 (20 pancreatic cancer patients and 20 healthy controls)), and the two sets were analyzed independently. We selected 10 peptide peaks showing a statistically significant difference between the cancer patients and controls (>2 fold difference, p < 0.0005 (Mann-Whitney U test), average peak intensity of >10 in either the cancer samples or the control samples) in both sets. We further selected 6 peaks of 412 m/z (RT 13.7 min), 546 m/z (8.3 min), 552 m/z (8.3 min), 827 m/z (8.3 min), 1141 m/z (29.0 min), and 1185 m/z (9.2 min) (supplemental Fig. S1 and Table S1) inspecting the 2DICAL reports with various two-dimensional views (Fig. 1B). The difference between cancer patients and controls was further validated in an independent set (Set 3, consisting of 5 pancreatic cancer patients and 4 healthy controls) obtained from another medical institution (Tokyo Medical University Hospital) (supplemental Fig. S2).

FIGURE 1.

Representative MS peak with significant difference between pancreatic cancer patients and controls. A, representative two-dimensional display of the entire set (>110,000) of MS peaks detected by 2DICAL with m/z values along the x-axis and retention time along the y-axis. B, peak at 552 m/z and 8.3 min displayed in various combinations of axes. Pancreatic cancer patients (Case) indicated in red and controls (Control) blue. Upper left, m/z and intensity axes with the indication of isotopic mass (light blue line and dot). Lower left, gray-scale intensity pattern of retention time (x axis) and sample (y axis). Upper right, sample and intensity axes (left) and a box-and-whisker diagram of pancreatic cancer patients and controls (right). Lower right, m/z and retention time axes with high (upper) and low (lower) intensities indicated by a red dot.

Target MS/MS Analysis for Peak Identification

Target MS/MS data were acquired from preparative LC-MS. The MS/MS spectra of the peaks of 552 m/z and 827 m/z matched the same ESSSHHP*GIAEFPSR sequence of fibrinogen α-polypeptide isoform α-E preproprotein (NP_000499/NP_068657) with the highest Mascot scores (supplemental Fig. S3 and not shown; * indicates a mismatch (described below)). These peaks were considered to be differently charged masses (triply and doubly charged, respectively) derived from the same peptide. The triple-charged 546 m/z peak is considered to be a mass with neutral loss of H₂O, because its appearance was almost identical to the peaks of 552 and 827 m/z. The peak of 1141 m/z matched another peptide sequence of fibrinogen α-polypeptide isoform α-E preproprotein TFP*GFFSPMLGEFVSETESR with the highest Mascot score (supplemental Fig. S4). No significant match was found with the 412 m/z peak despite its highly qualified MS/MS spectrum (data not shown), probably because of an unknown post-translational modification, a non-annotated gene sequence (26), or proteolysis. Qualified MS/MS spectra were not obtained from the peak of 1185 m/z. We noticed 16-dalton smaller MS peaks at different locations (547 m/z (8.5 min), 819 m/z (8.6 min), and 1133 m/z (30.0 min)) that completely matched the same amino acid sequences of fibrinogen α-polypeptide as the peaks of 552 m/z (8.3 min), 827 m/z (8.3 min), and 1141 m/z (29.0 min), respectively (Fig. 2 and data not shown). However, the intensity of the three 16-dalton-smaller MS peaks did not differ significantly between pancreatic cancer patients and controls (Fig. 2B).

FIGURE 2.

Modified and unmodified peptide fragments with the same amino acid sequences. A, peaks at 552, 827, 1141 m/z with significant difference between pancreatic cancer patients and controls. The axes are retention time and sample (top) and sample and intensity (bottom). B, peaks at 547, 819, and 1133 m/z matched to the above peaks of peptide fragment without modification.

Determination of the 16-Dalton Increase by High Resolution MS

To clarify the nature of the 16-dalton increase, the peptides of 827 and 819 m/z as well as 1141 and 1133 m/z were analyzed with a high resolution Orbitrap mass spectrometer. The difference between both the larger and the smaller pairs was 15.995 dalton, considered to be derived exclusively from the addition of one oxygen atom (Fig. 3, A and B). MS/MS revealed that the addition took place on the proline 565 and 530 residues (Fig. 3C and supplemental Fig. S5A). A 155.0808 m/z fragment observed in the high resolution MS/MS spectrum of the 819 m/z peak and a 171.0762 m/z fragment observed in the spectrum of the 827 m/z peak led to the exclusive identification of their chemical formulas as C₇H₁₁O₂N₂ and C₇H₁₁O₃N₂, respectively (Fig. 3D). Formulas C₇H₁₁O₃N₂ and C₇H₁₁O₂N₂ match hydroxyproline-glycine and unmodified proline-glycine, respectively.

FIGURE 3.

Determination of the 16-dalton increase by high resolution MS. A and B, high resolution MS spectra of the 827 and 819 m/z peaks (A) as well as the 1141 and 1133 m/z peaks (B) obtained with an Orbitrap mass spectrometer. C, MS/MS spectra of the 827, 1141, 819, and 1133 m/z peaks. D, 155.0808 m/z and 171.0762 m/z fragments exclusively identified as C₇H₁₁O₂N₂ and C₇H₁₁O₃N₂ with 5 ppm mass tolerance.

Detection of Prolyl 4-Hydroxylation of α-Fibrinogen

The physiologically stable oxidation of proline occurs exclusively at the carbon in the fourth position (supplemental Fig. S5B). We used Ganp (germinal center-associated nuclear protein) (27) transgenic mice to produce a monoclonal antibody (named 11A5) that reacts with a synthetic peptide ESSSHHP(O)GIAEFPSR (P(O), 4-hydroxyproline) (named HyP-ESS) but not with an unmodified synthetic peptide with the same amino acid sequence (ESS) (supplemental Fig. S6A). GANP mice can produce highly diverse antibodies and have been used with success to generate high affinity antibodies to various difficult antigens (18). We were unable to produce a monoclonal antibody with specificity for TFP(O)GFFSPMLGEFVSETESR (data not shown). Fibrinogen α-polypeptide is produced and secreted mainly by the liver. α-Fibrinogen with hydroxylation of its proline 565 residue (hereafter, αFG-565HyP) as well as 3 polypeptides (the α-, β-, and γ-chains) of fibrinogen were detected in the lysates (Fig. 4A) and supernatants (data not shown) of cultured hepatic cells and several hepatocellular carcinoma cell lines by immunoblotting with 11A5 monoclonal antibody. The 4-hydroxylation of proline is catalyzed by two types of enzymes: collagen-type and HIF1-type prolyl 4-hydroxylases (12, 28, 29). There are 4 collagen-type (P4HA1, P4HA2, P4HA3, and P4HB) and 3 HIF1-type (EGNL1, EGNL2, and EGNL3) prolyl 4-hydroxylase genes annotated in the human genome, but only knockdown of P4HA1 by siRNA inhibited the production of αFG-565HyP by KIM-1 cells (Fig. 4B and supplemental Fig. S7A), indicating the involvement of P4HA1 (EC 1.14.11.2) in the 4-hydroxylation of the proline 565 residue of α-fibrinogen, at least in this cell line.

FIGURE 4.

Detection of prolyl 4-hydroxylated α-fibrinogen by immunoblotting. A, lysates of normal hepatic cells (lane 1) and hepatocellular carcinoma KIM-1 (lane 2), Hep-3B (lane 3), SK-Hep-1 (lane 4), HLE (lane 5), Jhh-7 (lane 6), Hep-G2 (lane 7), HuH-7 (lane 8), and HuH-6clone5 (lane 9) cells were separated by SDS-PAGE and immunoblotted with anti-HyP-ESS and anti-fibrinogen (FG) antibodies. B, expression of the indicated genes was knocked in KIM-1 cells down by siRNA treatment (3 siRNAs for each gene). Forty-eight hours after transfection, the cell lysates were analyzed by immunoblotting with anti-HyP-ESS, anti-fibrinogen, and anti-β-actin (loading control) antibodies. NC, negative control (non-targeting RNA); NT, not treated. C, immunoblot analysis of plasma samples from pancreatic cancer patients and controls with anti-HyP-ESS and anti-fibrinogen antibodies.

Prolyl 4-Hydroxylated α-Fibrinogen in Clinical Samples

The plasma level of αFG-565HyP was increased in pancreatic cancer patients, but the levels of α-, β-, and γ-fibrinogen did not show any differences between pancreatic cancer patients and healthy controls (Fig. 4C). The levels of αFG-565HyP and α-fibrinogen were not significantly correlated (CC = 0.22) (supplemental Fig. S6, B and C). There was a significant correlation (CC = 0.81) between the intensity of the 827 m/z peak detected by 2DICAL and the level of αFG-565HyP determined by immunoblotting with 11A5 antibody (supplemental Fig. S7B), indicating the quantitative accuracy of 2DICAL. A competitive ELISA utilizing anti-HyP-ESS (11A5) monoclonal antibody was constructed (supplemental Fig. S6D), and the plasma level of αFG-565HyP was quantified in 685 individuals (Set 4) (Fig. 5). The plasma samples were collected prospectively from 7 medical institutions to ensure the absence of bias during the process of sample preparation. The ELISA assay showed high reproducibility with a median CV value of 0.079 among triplicates. There was a significant difference (p = 3.80 × 10⁻¹⁵, Mann-Whitney U test) in the plasma level of αFG-565HyP between 160 pancreatic cancer patients (2.26 ± 2.28 arbitrary units) and 113 healthy controls (0.91 ± 1.24) (Fig. 5A). The plasma level of αFG-565HyP was not elevated in patients with Stage IA (UICC, International Union Against Cancer) pancreatic cancer (p = 0.811), but patients with Stage IB or more advanced disease showed a significant increase of plasma αFG-565HyP (p = 2.99 × 10⁻² to 1.88 × 10⁻¹²) (Fig. 5B and supplemental Table S2). An elevated plasma level of αFG-565HyP was also observed in various cancers and chronic inflammatory disease. Patients with cancers of other organs (including the bile duct (p = 4.24 × 10⁻⁵), liver (p = 1.08 × 10⁻³), esophagus (p = 2.07 × 10⁻⁴), stomach (p = 5.95 × 10⁻⁴), and colon/rectum (p = 9.29 × 10⁻⁶)) as well as patients with chronic pancreatitis (p = 3.89 × 10⁻²) showed a significant increase in plasma αFG-565HyP (Fig. 5C and supplemental Table S3). Patients with benign pancreatic tumor/cyst (p = 0.216) or cholecystitis (p = 0.111) showed no significant difference from the controls.

FIGURE 5.

Quantification of plasma prolyl 4-hydroxylated α-fibrinogen. A, box-and-whisker diagram showing the different plasma levels of αFG-565Hyp in healthy controls (Cont, n = 113) and pancreatic cancer patients (Ca, n = 160). Boxes represent the median values and the 25–75 percentile range. Whiskers indicate the most extreme data point, which is no more than 1.5 times the interquartile range from the boxes. B, box-and-whisker diagram showing the plasma level of αFG-565Hyp in healthy controls (Cont, n = 113) and patients with stage-IA (n = 2), IB (n = 4), IIA (n = 5), IIB (n = 28), III (n = 41), and IV (n = 78) pancreatic cancer. C, box-and-whisker diagram showing the plasma level of αFG-565Hyp in healthy controls (Cont, n = 113) and patients with pancreatic ductal carcinoma (Du, n = 160), chronic pancreatitis (Pa, n = 12), benign pancreatic tumor or cyst (Be, n = 37), bile duct cancer (Bi, n = 25), cholecystitis (Cy, n = 22), hepatocellular carcinoma (He, n = 14), esophageal cancer (Es, n = 10), gastric cancer (St, n = 147), and colorectal cancer (Co, n = 145).

DISCUSSION

Plasma proteomics by liquid chromatography and mass spectrometry (LC-MS) has been a challenge because of the complexity and individual diversity of human samples. We developed a simple but robust method that enables the quantitative comparison of multiple LC-MS data. In this study, we identified 6 MS peaks whose intensity was significantly different between 38 cancer patients and 39 healthy controls (Figs. 1 and 2 and supplemental Figs. S1 and S2) among a total of 115,325 peaks derived from Con A-binding plasma glycoproteins. High resolution MS/MS analysis revealed that 4 of 6 peaks were derived from prolyl 4-hydroxylated plasma α-fibrinogen (Fig. 3). Artificial oxidation of peptides/proteins frequently occurs during the preparative procedures for MS analysis, especially during separation by SDS-PAGE. However, the plasma samples from cancer patients and healthy controls used in this study were collected, stored, and processed in an identical manner and were not separated by SDS-PAGE. We deliberately validated the native hydroxylation of the proline residue of plasma α-fibrinogen by immunoblotting and ELISA with a modification-specific monoclonal antibody (Figs. 4 and 5).

Prolyl hydroxylation is essential for the folding, secretion, and stability of the collagen triple helix (28, 29). Collagen has long been considered to be the only protein that is hydroxylated on its proline residues, but recently the von Hippel Lindau (VHL) tumor suppressor gene product-mediated degradation of HIF1α was revealed to be regulated by prolyl hydroxylation (12). Prolyl 4-hydroxylation regulates the stability of argonaute 2 protein (13). However, it is largely unknown which other proteins are prolyl-hydroxylated and how the modification regulates the function of proteins. We found that the collagen-type prolyl 4-hydroxylase P4HA1 is essential for the production of αFG-565HyP (Fig. 4B). Consistently, the consensus Xaa-Pro-Gly sequence of collagen (13, 30) was seen in the prolyl hydroxylation sites of α-fibrinogen (supplemental Fig. S5A). Prolyl-hydroxylated α-fibrinogen was produced in cultured hepatic cells and several hepatocellular carcinoma cell lines but not in pancreatic cancer cell lines (data not shown). Immunohistochemical study using antibody 11A5 showed that prolyl-hydroxylated α-fibrinogen existed at the inflammation site around the pancreatic cancer cells (data not shown). The modification change in plasma level may be determined by the production and consumption balance in the human body. Hydroxylation at proline 530 of α-fibrinogen was strongly correlated with αFG-565HyP (supplementary Fig. S7C). Multiple biological mechanisms may be involved in the regulation of prolyl-hydroxylated α-fibrinogen.

Post-translational modifications, such as glycosylation, phosphorylation, and oxidation, cause small differences in the molecular weight of proteins. Prolyl-hydroxylated peptides are 16 daltons larger than their unmodified counterparts, but this small change in molecular weight can readily be detected by 2DICAL as differences in the m/z values as well as the RT of the peptide peaks. The peaks derived from unmodified plasma fibrinogen fragments appeared in different locations (compare Fig. 2, A and B). Such modifications may be overlooked by MS/MS-based identification-oriented proteome approaches (31–34).

In this study, we were able to pinpoint the prolyl 4-hydroxylation of α-fibrinogen peptides in the large dataset of plasma samples (115,325 MS peaks × 231 LC-MS runs (77 cases in triplicate) = 27 million data points). Using a large independent validation cohort, newly constructed ELISA assay revealed the plasma level elevation of prolyl 4-hydroxylated α-fibrinogen in pancreatic cancer as well as other cancers and chronic inflammatory disease. Future studies will reveal the function of prolyl-hydroxylated α-fibrinogen and its regulation and clinical usage.