1Department of Medicine, National Jewish Medical and Research Center, Denver 80206; 5Biochemical Mass Spectrometry Facility, University of Colorado Health Sciences Center, Denver, Colorado 80262; 3Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232; 4Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of California, San Francisco, California 94143; and 2Department of Molecular and Structural Biology, Århus University, 8000 Århus C, Denmark
Submitted 3 September 2003 ; accepted in final form 16 January 2004
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ABSTRACT |
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bronchoalveolar lavage; proteomics; mass fingerprinting
ALI is a syndrome of complex pathogenesis that is a common complication of severe infections, trauma, and pneumonia (34, 64). The mortality of ALI has been reported to vary from 10 to 90% (4). One of the characteristics of ALI is severe dysfunction of the alveolar-capillary barrier. Normally, both endothelium and alveolar epithelium contribute to this barrier. In ALI, damage to this barrier (33, 53, 56) is likely responsible for a massive plasma protein efflux into the alveolar spaces that leads to hypoxemia and respiratory failure (3). ALI is characterized by the systemic activation of the acute-phase response leading to an elevation in acute-phase proteins in serum (16). The concomitant loss of the alveolar-capillary barrier means that these serum proteins are also seen in abundance in the air spaces (15). Until recently, technology had limitations that prevented a comprehensive description of these changes in ALI.
Both plasma and EF have been extensively studied to identify markers that predict the clinical course in ALI. Some of the many plasma markers include levels of angiotensin converting enzyme (18), clotting proteins (2, 36, 52), antioxidants (35), surfactant proteins (8, 14), cytokines (10), coagulation markers (45, 63), and adhesion molecules (47). Distal air space concentrations of antioxidants (5, 37), surfactant proteins (8), collagen fragments (9), von Willebrand factor antigen (62), human type I (HTI)56 a human alveolar epithelial type I cell antigen (40), and platelet-activating factor (38) have also been shown to correlate with clinical outcomes in ALI.
The strength of proteomics as a discovery tool is that it allows previously unrecognized associations to be identified. To date there have been no reports of the application of this strategy to EF in ALI. We anticipated that a proteomic approach to studying ALI would confirm that the alveolar capillary barrier is deficient in ALI and that we would identify both changes in relative protein intensity and protein isoforms that might not be detected by other techniques. It is our thesis that gene products are extensively modified posttranslationally to yield a complex array of protein products with subtle structural differences but potentially distinct biological activity. These isoforms are not discernible by genomic or antibody-based strategies, yet they are likely to yield important information regarding protein function.
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MATERIALS AND METHODS |
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All subjects were studied under protocols approved by the Institutional Review Board at the University of Colorado Health Sciences Center, National Jewish Medical and Research Center, and by the Committee on Human Research of the University of California, San Francisco, in accordance with guidelines recommended by the National Institutes of Health. The normal subjects were nonsmokers, ages 2235 (see Table 1). The subjects were sedated with midazolam, topically anesthetized with lidocaine, and then had BALF sampled by transnasal bronchoalveolar lavage with four sequential 60-ml volumes. The return was pooled into one sample. Venous blood was obtained by antecubital venipuncture. ALI patients were endotracheally intubated subjects who fulfilled the criteria for ALI (4). EF from ALI subjects was sampled immediately after endotracheal intubation by direct aspiration through a catheter wedged in a distal airway as previously described (67). Occasionally, a known volume of normal saline was added to samples to decrease viscosity. Blood samples were obtained in heparinized glass tubes, placed on ice, and centrifuged immediately at 4°C.
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One tablet of Complete Protease Inhibitor Cocktail (Roche, Mannheim, Germany), an inhibitor of a wide variety of serine proteases, cysteine proteases, and metalloproteases, was added to fresh BALF and EF samples. To dilute samples with high protein concentrations, we added one volume of normal saline to EF samples from ALI patients. The fluid was spun (1,500 g, 10 min, 4°C) to remove cellular debris. The cell-free supernatants were frozen at 80°C. Blood samples were spun (1,500 g, 10 min, 4°C), and the plasma was stored at 80°C. All reagents were supplied by Sigma Chemical (St. Louis, MO) unless otherwise noted.
Two-Dimensional Gel Electrophoresis
Total protein in plasma, BALF and EF was determined by the bicinchoninic acid assay and employed bovine albumin standards (Pierce, Rockford, IL). Samples were dialyzed against deionized water [24 h, room temperature (RT)] to remove salts from sample preparation and were then quick frozen in liquid nitrogen. To concentrate the bronchoscopy samples, we centrifuged 15 ml under reduced pressure until the volume reached 100200 µl. In preliminary studies we compared several additional protein isolation methods such as acetone precipitation, trichloroacetic acid precipitation, and protein concentrators (Amicon) to concentrate proteins from dilute BALF; however, none was superior to that described above. Although this approach allowed the identification of hundreds of spots, it is limited to water-soluble proteins with a isoelectric point (pI) 310 and molecular masses of 10200 kDa. For each sample of plasma, BALF, and EF, 500 µg of protein were reconstituted in rehydration buffer {8 M urea, 2% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 0.5% pH 310 immobilized pH gradient (IPG) buffer, 45 mM dithiothreitol (DTT), and a trace of bromphenol blue} to a final volume of 350 µl. The samples were incubated (RT, 30 min) and then used to rehydrate 17-cm IPG strips (Bio-Rad) with a pH 310 linear gradient. The strips were actively rehydrated (50 V, 24 h, 20°C) in a Protean isoelectric focus (IEF) Cell (Bio-Rad). First-dimension IEF was carried out at 20°C in a Protean IEF Cell by the following protocol: 250 V for 15 min, 10,000 V for 3 h, then overnight at 10,000 V for 60,000 V-h followed by a 500-V hold. After IEF, strips were equilibrated by agitating for 10 min in 50 mM Tris·HCl, pH 8.8, 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) sodium dodecyl sulfate (SDS), and 650 mM DTT and then agitating for 10 min in 50 mM Tris·HCl, pH 8.8, 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, and 1.27 M iodoacetamide. The strips were next loaded onto a 20 x 20-cm 12% polyacrylamide gel and subjected to an electric field in a Protean Plus Dodeca cell [Bio-Rad; 4°C at a constant 10 mA per gel, 21 h in a running buffer containing 25 mM Tris, 192 mM glycine, and 0.1% (wt/vol) SDS]. After electrophoresis, proteins were visualized by Sypro Ruby staining (Molecular Probes, Eugene, OR).
Identification of Protein Spots
Sypro Ruby-stained spots were manually excised from gels under an ultraviolet light box. Spots were excised if they appeared in at least two gels, and we attempted to identify as many spots as possible from all the gels. In some cases identical spots were excised from multiple gels and combined to yield more material. The gel spots were subjected to reduction and alkylation by repeated dehydration with acetonitrile between washes (10 mM DTT/0.1 M NH4HCO3, 55 mM iodoacetamide/0.1 M NH4HCO3, and 0.1 M NH4HCO3). The gel spots were rehydrated for digestion with 0.1 µg of sequencing-grade modified trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate and incubated overnight at RT. The peptide samples were then cocrystallized with matrix (-cyano-4-hydroxycinnamic acid) on a sample plate (Applied Biosystems, Foster City, CA) using 1 µl of matrix and 1 µl of sample. An Applied Biosystems Voyager-DE STR, operating in delayed reflector mode with an accelerated voltage of 20 kV, was used to generate peptide mass maps. All samples were externally calibrated with peptide standards, and the resulting mass spectra were internally calibrated with trypsin autolysis products. Monoisotopic peptide masses were assigned and then used in Mascot database searches (24). A mass fingerprint was considered a match if the score was
70.
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RESULTS |
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The normal subjects were significantly younger (25 ± 5 years) than the ALI patients (53 ± 3 yr, P < 0.001; Table 1). The in-hospital mortality in the ALI group was 25%, similar to that previously reported (54, 64, 68). The causes of ALI were sepsis (n = 4), pneumonia (n = 6), aspiration (n = 4), and liver failure (n = 2). Comorbid illnesses were radiation pneumonitis (n = 2), asthma (n = 1), chronic renal failure (n = 1), metastatic renal cancer (n = 1), and cardiac transplantation (n = 1). Compared with normal subjects, plasma protein was depressed in ALI patients (8.6 ± 0.6 vs. 5.8 ± 0.4 g/dl, P < 0.01). Because bronchoalveolar lavage results in dilution of air space fluid, the protein content from normal subjects was lower than in ALI patients (66 ± 20 vs. 46,270 ± 6,887 µg/ml, P < 0.01).
BALF and Plasma Proteome
A typical 2-DE gel from the BALF of normal subjects and the EF of ALI patients had about 300 spots detected by Sypro Ruby staining. A 2-DE gel of the BALF of a healthy, nonsmoking 28-year-old woman is shown in Fig. 1; the 2-DE images from 11 other healthy volunteers were similar (data not shown). From the plasma, BALF, and EF samples analyzed by 2-DE, a total of 158 spots were identified by peptide mass fingerprinting. Table 2 summarizes all the protein identifications using data obtained from all patients. A total of 72 unique proteins were identified among all samples. Although many of these identifications have been previously reported in BALF (20, 31, 32, 39, 41), few studies have compared the EF proteome to the plasma proteome or have remarked on protein isoforms. To make these comparisons, we examined plasma and BALF obtained simultaneously from normal subjects.
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The expression profiles for the healthy volunteers were globally similar; however, from individual to individual there was some variability in spot intensity (e.g., immunoglobulin spots 42 on Fig. 1 vs. 2A). For other proteins, multiple isoforms were evident, predominantly in the BALF. For instance, in BALF of all normal subjects there were seven distinct spots for surfactant protein A (SP-A) (Fig. 1, spots 5965). For these spots, the apparent molecular mass was 710 kDa higher than the calculated molecular mass that is based on amino acid sequence alone and does not consider posttranslational modifications. Furthermore, as the apparent (observed) molecular mass of each spot increased, the observed pI became more acidic by 0.1. There was no SP-A detected in the plasma, although a more sensitive antibody-based assay is capable of detecting this protein in plasma (8).
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Although there were only a few proteins that were identified exclusively in the BALF, there was a substantial difference in intensity between the plasma and BALF. Albumin, haptoglobin, IgG, fibrinogen ( and
), apolipoprotein, clusterin-sulfated glycoprotein-2, transferrin, retinol binding protein, and transthyretin all stained more intensely in the plasma compared with the BALF. In normal subjects there were many proteins that were identified in the plasma but not BALF (e.g., apolipoprotein, orosomucoid) or BALF but not plasma (e.g., SP-A, IgJ)
EF and Plasma Protein Profile in Patients With ALI
Representative 2-DE images of plasma and EF from a patient with ALI are shown in Fig. 3. The qualitative differences among the protein profiles of the 16 ALI patients were similar to these examples and can be grouped into three broad categories: first, proteins with increased relative intensity compared with normal subjects; second, proteins with decreased relative intensity compared with normal subjects; and third, proteins having undergone posttranslational modifications, i.e., modified in a manner that changed either or both the molecular weight and the pI.
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Decreased relative protein intensity in ALI patients.
SP-A, which was seen in the BALF of all normal subjects, was identified in only one patient with ALI. Similarly, 1-antitrypsin (
1-AT), which was identified in all of the normal subjects, was evident in only half the ALI patients. The observed and calculated pI and molecular mass of SP-A were similar; however, a variant was identified as the COOH-terminal portion of the protein (amino acids 266394). This spot was evident in the EF of six ALI patients but only two normal subjects.
Modified expression.
Several proteins identified in the EF of ALI patients were truncated and displayed observed molecular masses that were significantly less than expected based on the amino acid sequence alone. For example, for haptoglobin, there were five isoforms evident with molecular masses of 45 kDa, but with distinct pIs ranging from 5.3 to 5.7. These isoforms were observed at a slightly higher molecular mass than calculated, and the protein was slightly more acidic than predicted. These forms were evident in the plasma but not BALF of normal patients and ALI patients. We also identified three isoforms of haptoglobin with an observed molecular mass of 22 and pIs in the range 5.46.4. This molecular mass is less than half that predicted, and the coverage maps of these proteins included only the NH2-terminal portion of the protein. These lower-molecular mass haptoglobins were identified in the EF of all but two ALI patients, but they were not evident in samples collected from normal subjects. Similarly, two isoforms of complement component 3 were identified. The coverage maps of these lower-molecular mass forms represented peptides derived from only the COOH-terminal portion of the protein. These two forms were only evident in the EF of ALI patients, but in none of the normal subjects.
For some proteins additional isoforms were evident in both the plasma and BALF. For instance, in the plasma and BALF of all normal subjects there was only one form of orosomucoid (also called 1-acid glycoprotein) present; however, an additional isoform that stained strongly was evident in the plasma and the EF of all but one of the ALI patients (Fig. 4). These two isoforms were distinguished by a small decrease in pI, but there was no observable change in molecular mass. The coverage maps did not indicate differences in primary amino acid sequence.
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DISCUSSION |
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Although the proteomics approach to studying clinical problems is in its infancy, DNA microarrays were in a similar stage of development several years ago, and now their use is widespread. Microarrays have been used to improve the understanding of the molecular pathogenesis of disease and to predict clinical outcomes based on improved phenotyping. For instance, we previously showed that superoxide mimetics attenuates ischemia-reperfusion injury of the brain (50), and we employed microarrays to show that this effect was associated with diminished activation of inflammatory pathways (6). Microarrays have also been used to identify genes that will predict breast and esophageal cancer responses to chemotherapy (27, 42). DNA microarrays have been used to predict the clinical outcome of breast cancer (60), B-cell lymphomas (46, 51), and acute renal allograft rejection (48). One feature that distinguishes the proteomics approach from DNA microarrays is the ability to identify distinct protein isoforms that may be critical in determining disease pathogenesis. Our results demonstrate that a single gene product may be present in many isoforms and that some of these isoforms are associated with ALI. A limitation to the 2-DE proteomics approach is that it is not easy to quantify all changes in relative protein expression among a large number of samples. For this and many other reasons, the proteomics approach may be complementary to microarray studies.
Most of the proteins identified in the EF of ALI patients were plasma proteins, consistent with the theory that ALI lung injury is characterized by increased permeability of pulmonary capillaries; however, there were additional features of the ALI protein expression profile that indicate modified protein synthesis and/or degradation. Some of these changes include the apparent loss of alveolar synthetic function (e.g., SP-A), truncation of proteins (e.g., evidence of proteolysis), and subtle changes in protein expression (e.g., orosomucoid). The identity and significance of these posttranslational modifications require additional study and may shed new light into the pathogenesis of ALI.
The protein profiles seen in the ALI subjects of this investigation have remarkable similarities with those reported by previous investigators. For instance, Wattiez et al. (66) studied the BALF proteome from five healthy subjects, three patients with idiopathic pulmonary fibrosis (IPF), and two patients with HP. The investigators were able to detect semiquantitative relative decreases in the intensity of SP-A in patients with HP and IPF but relative increase in intensity of proteins such as transferrin, transthyretin, 1-AT, and immunoglobulin. The IPF and HP patients also demonstrated numerous spots corresponding to the NH2-terminal sequences of haptoglobin. This study did not extend the pI range to 3.0 and thus did not identify strongly acidic proteins such as orosomucoid. In another study of eight healthy children and 17 children with cystic fibrosis, there was increased intensity of
1-AT and lower-molecular-weight isoforms of SP-A (61). In an analysis of BALF collected from seven healthy children undergoing diagnostic operations and 10 children with malignancies, fever, and chest infiltrates, the sick children were noted to have large increases in intensities of
1-AT and hemoglobin, decreases in transthyretin, but no changes in SP-A, transferrin, or immunoglobulins (39). Therefore, many of the changes that were observed in the ALI subjects of this study are consistent with those reported in studies of other lung diseases.
Contrasting the BALF and plasma protein expression profile in normal subjects with the EF and plasma protein profile in ALI patients underscores several changes that contribute to the pathology of ALI. First, there is loss of size selectivity of the alveolar-capillary barrier such that high-molecular mass proteins including albumin, transferrin, and immunoglobulin leak from plasma into the air spaces (25). Recent studies of ceruloplasmin and albumin in ALI patients have implicated plasma exudation as the source of these proteins (3). In the current investigation the BALF from normal subjects had relatively lower intensity of high-molecular mass proteins such as albumin, immunoglobulin, and transferrin compared with low-molecular mass proteins such as transthyretin. The ALI patients had a relative increase in the intensity of high-molecular mass proteins in their EF. This confirms the integrity and size selectivity of the alveolar-capillary barrier in normal subjects compared with ALI patients. Second, alveolar type II cell function may be impaired during ALI. This observation would account for the relative decreases in EF SP-A that were observed in this and other studies (8, 21). Third, there may be enhanced proteolytic activity in the EF of ALI patients (11, 49, 64). Interestingly, this did not appear to affect all proteins equally; for instance, haptoglobin and 1-AT had observed molecular masses that were significantly less than calculated, yet other proteins remained intact. Fourth, ALI and other inflammatory diseases have been associated with increases in acute-phase proteins (19). We found that there are relative increases in intensity of several acute-phase proteins (such as serum amyloid A and
1-AT) in both the EF and plasma of ALI patients. These findings are consistent with results from previous investigations that examined the association between ALI and individual acute-phase proteins such as ceruloplasmin, albumin, and
2-macroglobulin (3) and surfactant proteins (8), as well as a previous study of crossed immunoelectrophoresis of sera from 15 normal subjects and 10 ALI patients (16).
Orosomucoid is another acute-phase protein in which a subtle modification is associated with ALI. This modification would not have been evident if we had used microarray or antibody (e.g., ELISA, Western blotting, or radioimmunoassay) strategies. The identification of an isoform of orosomucoid that is associated with ALI highlights the unique strengths of the proteomics approach. Orosomucoid is a highly sialylated, acidic protein that is synthesized primarily by hepatocytes but also by epithelial and endothelial cells of nonhepatic organs (17, 23, 55, 58). There are three genes encoding for orosomucoid (AGP-A, AGP-B, and AGP-B'). ORM1 is the protein product of AGP-A; ORM2 is the protein product of AGP-B and AGP-B' (12). Because of the close homology between ORM1 and ORM2, we were not able to distinguish between the two by MALDI-TOF MS identification. However, previous reports indicate that most orosomucoid detected in tissues and plasma is ORM1 and its glycosylated variants (17). A putative role for the variant isoform is that it is immunomodulatory because of its altered carbohydrate composition (17). For instance, the sialyl LewisX form of orosomucoid reduces neutrophil inflammation in the lung by binding to the cell adhesion molecules E-selectin and P-selectin (28, 44). A previous study reported a cathodic shift in orosomucoid obtained from BALF in ALI patients, but the significance of this is unclear (15). Additional investigations are needed reveal the nature of this variant and its significance in ALI.
We should stress that although the proteome is defined as the complete protein complement of a biologic sample, existing methods limit analysis to only a subset of the proteome. For example, sample preparation for gel electrophoresis preselects for a subset of the proteome based on pI, molecular mass, solubility, etc. Consequently, hydrophobic proteins are missed by this analysis. Second, not all proteins, particularly the low-molecular mass proteins, are readily identifiable using the combination of 2-DE and MALDI-TOF MS. Furthermore, low-abundance proteins (e.g., cytokines) are usually not detected unless analyte-concentration steps are incorporated. For example, we were not able to detect SP-A by 2-DE in the ALI patients but others have been able to detect SP-A by enzyme-linked immunosorbent assays (8). Thirdly, there was some intersubject variability in relative protein intensity among normal subjects. Because the bronchoscopy method was standardized, it is unlikely that the bronchoscopy technique accounts for the majority of variability. Instead, the intersubject variability in protein profile most likely reflects biologic diversity. Fourthly, we were able to neither identify nor quantify all protein spots. To identify specific proteome patterns in subsets of ALI (e.g., sepsis, aspiration), improvements in automated spot quantitation and identification should be adopted, and larger patient populations will be required. Finally, the factors that determine protein expression in the plasma and lung have not yet been fully identified. Thus we cannot exclude the possibility that variables such as sampling technique, age, and mechanical ventilation confound this interpretation.
In conclusion, this study demonstrates the potential of proteomics to study the pattern of air space proteins in ALI patients. One significant advantage is the ability to detect subtle posttranslational modifications that would not be evident by other methods (e.g., orosomucoid and haptoglobin). Our work confirms that plasma proteins are abundant in the distal air spaces of ALI patients but also identifies groups of proteins that have relative increased or decreased intensity, have lower than expected molecular masses, or novel isoforms arising from subtle posttranslational modifications. Confirmation of these findings using refined subsets of proteins and methods that more precisely quantify these observations will be required. Furthermore, prospective and longitudinal studies using larger samples size, well-defined disease states and tightly matched control groups will be needed to evaluate and guide individual therapeutic treatments in the future.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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