Proteomic Analysis of Human Neutrophil Granules*,S

George Lominadze{ddagger}, David W. Powell§, Greg C. Luerman{ddagger}, Andrew J. Link§, Richard A. Ward and Kenneth R. McLeish{ddagger},||,**

From the Departments of Medicine and {ddagger} Biochemistry and Molecular Biology, University of Louisville and the || Veterans Affairs Medical Center, Louisville, Kentucky 40202 and the § Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.


Stimulated exocytosis of membrane-bound compartments plays a critical role in converting inactive, circulating neutrophils to fully activated cells capable of directed migration, phagocytosis, and killing of microbes (1). Four subsets of membrane-bound compartments exist in neutrophils: secretory vesicles, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules (1, 2). Secretory vesicles are generated by endocytosis, resulting in membranes rich in receptors, signaling proteins, and adhesion molecules, whereas their lumen contains plasma (2). Azurophil, specific, and gelatinase granules contain various enzymes and host defense proteins in their luminal spaces, whereas their membranes contain receptors, signaling proteins, adhesion molecules, and enzymes (2, 3). The targeting of proteins to individual granule compartments is determined by the timing of protein synthesis during myeloid progenitor cell differentiation, not by granule-specific sorting (4). Thus, granule proteins that are synthesized at a given stage of cellular differentiation will be localized to the same type of granule (5, 6). The overlap between the timing of synthesis of different granular proteins gives rise to some overlap of contents among different granule subsets (6, 7).

The extent of mobilization of the three types of granule depends on the stimulus intensity, whereas the order of mobilization is fixed, a process termed graded exocytosis (8, 9). Gelatinase granules are most easily mobilized followed by specific granules and then azurophil granules. Graded exocytosis results in stepwise addition of granule membrane proteins to the plasma membrane and stepwise release of granule luminal contents into the extracellular space. The controlled exocytosis of neutrophil granules allows sequential acquisition of functional responses and targeted delivery of toxic granule proteins, thereby reducing damage to normal host tissue.

In addition to graded granule release, neutrophil exocytosis is distinguished by two other attributes. The first is a lack of spatial organization of granules (1012). The random distribution of granules in the cytosol of circulating neutrophils suggests that graded exocytosis requires a mechanism to discriminate among granule subsets. The second attribute is compound exocytosis, the fusion of two or more granules prior to their fusion with the plasma membrane (13). Compound exocytosis requires recognition of granule subsets as target membranes to allow for homotypic or heterotypic fusion. Homotypic fusion enhances the localized delivery of granule contents. Heterotypic fusion allows processing of certain granule constituents into active forms by proteolytic cleavage (14).

The molecular mechanisms that control exocytosis of neutrophil granules are poorly defined. Additionally the full complement of functional changes resulting from exocytosis of different granule subsets remains to be identified. A major reason for this limited understanding of neutrophil exocytosis is an incomplete identification of membrane and luminal proteins of each granule subset. To address this problem, we performed proteomic profiling of the components of azurophil, specific, and gelatinase granules from human neutrophils. Two different methods for granule protein identification were applied. One used two-dimensional gel electrophoresis (2DE)1 followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins. In the other, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets. Additionally optimal methods for protein identification differed among granule subsets based on their physical properties and luminal components.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil Isolation
Neutrophils (8 x 108 cells) were isolated from healthy donors using plasma-Percoll gradients as described by Haslett et al. (15). Trypan blue staining revealed that at least 97% of cells were neutrophils with >95% viability. After isolation neutrophils were suspended in Krebs-Ringer phosphate buffer (pH 7.2) at 4 x 107 cells/ml and treated with 10 µM diisopropyl fluorophosphate for 10 min on ice to inhibit proteases (16). The Human Studies Committee of the University of Louisville approved the use of human donors.

Subcellular Fractionation for Granule Enrichment
Neutrophil granules were enriched by centrifugation on a three-layer Percoll density gradient as described by Kjeldsen et al. (17). Briefly isolated neutrophils (4 x 107/ml) were resuspended in disruption buffer containing 100 mM KCl, 1 mM NaCl, 1 mM ATPNa2, 3.5 mM MgCl2, 10 mM PIPES, and 0.5 mM PMSF and disrupted by nitrogen cavitation at 380 p.s.i. and 4 °C. The cavitate was collected and supplemented with 1.5 mM EGTA, and nuclei and intact cells were removed by centrifugation at 500 x g for 5 min. The postnuclear supernatant was layered onto a discontinuous Percoll gradient formed from three 9-ml layers of Percoll prepared in a buffer containing 100 mM KCl, 3 mM NaCl, 1 mM ATPNa2, 3.5 mM MgCl2, 1.25 mM EGTA, 10 mM PIPES, and 0.5 mM PMSF to achieve final densities of 1.050, 1.090, and 1.120 g/ml. The gradient was centrifuged at 37,000 x g for 30 min in an SS-34 fixed angle rotor in a Sorvall RC-5B centrifuge. The separated granule fractions were recovered from the gradient interfaces by aspiration, and Percoll was removed by ultracentrifugation of each granule subset at 100,000 x g for 90 min.

Sample Preparation for 2DE
Preparation of Whole Granules—
Whole granule fractions were resuspended in 4 ml of disruption buffer and centrifuged at 100,000 x g for 20 min to obtain a solid pellet. Buffer was removed by aspiration, and the pellets were washed briefly with deionized water to remove residual salt.

Fractionation of Granule Proteins—
To separate granule proteins into membrane and luminal fractions, granules were resuspended in 10 volumes of 0.1 M sodium carbonate, sonicated at high setting for 5 s, and subjected to three freeze-thaw cycles each followed by sonication (18). Disrupted granules were incubated on ice for 30 min, and carbonate-washed membranes were pelleted at 100,000 x g. The supernatant from the carbonate wash was concentrated by ultrafiltration through 1-kDa-cutoff centrifugal devices (Microsep Omega, Pall, East Hills, NY), and the retentate proteins were precipitated using chloroform-methanol desalting-precipitation (19). The precipitate was dried on room air and subjected to 2DE.

To separate granule proteins based on differential solubility in ammonium sulfate solution, whole granules were solubilized in 3 volumes of lysis buffer containing 50 mM Tris, pH 7.2, and 2% Triton X-100 and centrifuged at 20,000 x g to remove insoluble proteins. The Triton X-100-insoluble pellet was subjected to 4–12% acrylamide gradient SDS-PAGE and MALDI MS analysis, while the cleared lysate was used for fractionation by ammonium sulfate precipitation of proteins. One volume of 100% saturated ammonium sulfate solution was added to the lysate. The resulting precipitate was pelleted (subfraction one), and supernatant was removed. Solid ammonium sulfate was added to the supernatant until saturation, and the protein precipitate was pelleted (subfraction two). Precipitated protein pellets were redissolved in the lysis buffer and subjected to chloroform-methanol desalting-precipitation (19).

Protein Separation by 2DE and Identification by MALDI-TOF MS
Whole granules and granule subfractions were dissolved in 160 µl of 7 M urea, 2 M thiourea rehydration buffer (Genomic Solutions, Ann Arbor, MI). Proteins in all samples were separated by 2DE using non-linear pH 3–10 IPG strips for the first dimension and 4–12% gradient acrylamide gels for the second dimension (Invitrogen). The gels were visualized by colloidal Coomassie staining. Proteins were excised and in-gel trypsin-digested, and the resulting peptides were analyzed by MALDI-TOF MS using the thin film sample preparation method as described previously (20, 21). Protein identification was carried out by searching peptide spectra against the National Center for Biotechnology Information (NCBI) database using the Mascot web-based search engine. The search parameters used were: taxonomy, Homo sapiens; allowed error, 150 ppm; fixed modification, carbamidomethylation; variable modification, methionine oxidation; mass values, MH+; and maximum allowed missed cleavage, 1.

Protein Identification by 2D HPLC ESI-MS/MS
Granule membranes obtained following treatment with 0.1 M sodium carbonate, as described above, were washed in 50 mM ammonium bicarbonate and then resuspended by sonication in 300 µl of 50 mM ammonium bicarbonate. Trypsin digestion was performed by addition of 20 ng/ml trypsin to the suspension, and samples were incubated on a rotator overnight at 37 °C. Residual particulate material was removed by centrifugation at 100,000 x g, and the trypsin-generated mixture was analyzed using an approach that combined two-dimensional microcapillary HPLC with ESI-MS/MS (22). All tandem spectra were searched against H. sapiens open reading frame database (human.nci) using the SEQUEST algorithm (23). For singly charged peptides, spectra with a cross-correlation score of greater than 1.5 were retained, whereas for multiply charged peptides, spectra with a cross-correlation score of greater than 2 were retained (24). The analysis was repeated three times for each granule subset, and only proteins identified in at least two of three experiments were assumed to be present on granules.

Quantitation of Granule Membranes and Western Blot Analysis of Granules
Whole granule fractions were resuspended in 2 ml of disruption buffer and centrifuged at 100,000 x g for 20 min to obtain a solid pellet. Buffer was removed by aspiration, and the pellets were resuspended in 100 µl of disruption buffer. Sample volume was brought up to 1 ml by water and preincubated at 37 °C. To quantitate phospholipid bilayers, TMA-DPH (Molecular Probes, Eugene, OR) was added at a final concentration of 1 x 10–7 M, and fluorescence intensity was monitored continuously at excitation of 350 nm and emission of 430 nm for 20 s on a Hitachi 4500 fluorescence spectrometer. Proteins associated with equal amounts of membrane from each granule subset were loaded onto a gel for SDS-PAGE. Proteins were separated, transferred to nitrocellulose membrane, and probed with an anti-actin antibody.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prior to subjecting granules to proteomic analysis, the purity of each of the three granule subsets was determined. Granule subsets were analyzed by Western blotting for CD66b (specific granule marker) and by ELISA for gelatinase and myeloperoxidase (MPO), markers for gelatinase and azurophil granules, respectively (Fig. 1). The Western blot showed that CD66b was present in the specific granule fraction but essentially absent in gelatinase granule and azurophil granule fractions. ELISA for gelatinase showed that 75% of total gelatinase was present in gelatinase granules, 20% was in specific granules, and 5% was in azurophil granules, whereas 73% of MPO was in azurophil granules, 20% was in specific granules, and 7% was in gelatinase granules. This distribution of granule markers was similar to that reported by Kjeldsen et al. (17, 25). Thus, optimal levels of granule subset enrichment were obtained.



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FIG. 1. Assessment of purity of granule fractions. Granule fractions separated by centrifugation on Percoll gradients were analyzed for the content of CD66b (specific granule marker), gelatinase (gelatinase granule marker), and myeloperoxidase (azurophil granule marker). A, Western blotting analysis of CD66b content in granule fractions. B, ELISA for gelatinase. C, ELISA for myeloperoxidase.

 
The initial analysis of granule subset proteomes was carried out by subjecting whole granules to 2DE. Fig. 2A shows the separation of gelatinase granule proteins. A total of 38 proteins were identified by peptide mass fingerprint analysis of MALDI MS spectral data. Thirty of these proteins were cytoskeletal and luminal proteins. The 2D gels of specific granule proteins were dominated by large amounts of luminal proteins, such as lactoferrin and lipocalin (NGAL), which were present in such high abundance that proteins focused poorly (Fig. 2B). A total of 26 proteins were identified by peptide mass fingerprinting. The only cytoskeletal protein identified was actin (Fig. 2B). Separation of azurophil granule proteins by 2DE revealed large amounts of highly basic proteins, such as myeloperoxidase, that focused poorly, leading to extensive smearing on the basic end of the gel (Fig. 2C). Only eight proteins were identified from the azurophil granule gel.



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FIG. 2. 2D gels of whole granules. Neutrophil granules were subjected to 2DE, gels were stained with colloidal Coomassie Blue dye, the spots were excised and in-gel digested with trypsin, and the resultant peptides were analyzed by MALDI-TOF MS. A, gelatinase granules. B, specific granules. C, azurophil granules.

 
To address the problem of overabundance of luminal proteins, granule proteins were fractionated by one of two methods prior to their separation by 2DE. First, the luminal contents of granules were separated from membranes by lysing the granules in 0.1 M sodium carbonate, pH 11. At high pH luminal cationic proteins lose their positive charge and dissociate from negatively charged matrix (18, 26, 27). Additionally alkaline sodium carbonate treatment has been reported to remove the actin cytoskeleton from neutrophil membranes (28). To determine what proteins were removed from granule membranes, carbonate-soluble proteins were also subjected to 2DE. Second, differential solubility of proteins in ammonium sulfate solutions was applied to granule proteins prior to 2DE. Whole granules were solubilized in a buffer containing Triton X-100, and proteins were separated based on precipitation at different concentrations of ammonium sulfate. The first subfraction contained proteins precipitated in a 20% ammonium sulfate solution, whereas the second subfraction contained the proteins soluble in 20% ammonium sulfate but insoluble in 100% ammonium sulfate.

Fractionation of gelatinase granules with sodium carbonate prior to 2DE resulted in identification of 26 proteins of which eight proteins were not detected on whole granule gels (Fig. 3, A and B). On the other hand, 20 proteins identified by 2DE of whole granules were not observed on either carbonate-washed membrane or carbonate-soluble protein gels. Most of these proteins were below 30 kDa, suggesting that low molecular weight proteins were lost during fractionation. Fig. 3, C and D, shows 2DE separation of ammonium sulfate-precipitated proteins from gelatinase granules. This fractionation allowed identification of 38 proteins, 22 of which were not previously identified. Use of ammonium sulfate precipitation also identified all but three proteins detected following fractionation with sodium carbonate. Ten proteins seen in whole gelatinase granule gels were not detected after either fractionation method, indicating that protein fractionation is complementary to analysis of intact gelatinase granules. A total of 62 proteins were identified from gelatinase granules by 2DE protein separation and MALDI MS (Table I).



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FIG. 3. 2D gels of gelatinase granule proteins fractionated by carbonate lysis and by ammonium sulfate precipitation of Triton X-100-solubilized proteins. Shown is fractionation of gelatinase granule proteins by carbonate lysis (A and B) and by ammonium sulfate precipitation (C and D). A, a 2D gel of proteins that remained on the membrane after granule lysis in 0.1 M sodium carbonate buffer. B, proteins that were solubilized in 0.1 M sodium carbonate. C, proteins precipitated in 20% ammonium sulfate solution. D, proteins that remained in solution in 20% ammonium sulfate but were precipitated in 100% ammonium sulfate solution.

 

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TABLE I Proteins identified from 2D gels by MALDI-TOF MS

Eighty-seven proteins were identified from all 2D gels of neutrophil granules. The table lists the gene and protein designation, granule type, number of peptides matched, the percent protein coverage by these peptides, and the calculated molecular weight search (MOWSE) score for each protein. SOD, superoxide dismutase; GE, gelatinase; SP, specific; AZ, azurophil; VDAC, voltage-dependent anion channel; ARP, actin-related protein; LSP1, lymphocyte-specific protein 1; BP, binding protein; TOLLIP, Toll-interacting protein; Arc, ARP-related complex; Grp, glucose regulated protein; Hsp, heat shock protein.

 
Fractionation of specific granule proteins with sodium carbonate resulted in identification of more proteins than extraction by ammonium sulfate precipitation. Sodium carbonate treatment prior to 2DE resulted in identification of 51 proteins from membranes and 27 proteins in the supernatant of which 42 proteins were not detected on gels from whole specific granules (Fig. 4, A and B). Ammonium sulfate precipitation allowed identification of 45 proteins, only 10 of which were not seen on gels from whole granules. Ammonium sulfate precipitation did not lead to identification of any protein not identified after fractionation with sodium carbonate (Fig. 4, C and D). Collagenase and cathepsin X were identified on the whole specific granule gels but not after extraction by sodium carbonate. A total of 70 proteins were identified from specific granules (Table I).



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FIG. 4. 2D gels of specific granule proteins fractionated by carbonate lysis and by ammonium sulfate precipitation of TX-100-solubilized proteins. Shown is fractionation of specific granule proteins by carbonate lysis (A and B) and by ammonium sulfate precipitation (C and D). A, a 2D gel of proteins that remained on the membrane after granule lysis in 0.1 M sodium carbonate buffer. B, proteins that were solubilized in 0.1 M sodium carbonate. C, proteins precipitated in 20% ammonium sulfate solution. D, proteins that remained in solution in 20% ammonium sulfate but were precipitated in 100% ammonium sulfate solution.

 
Fractionation of azurophil granule proteins by sodium carbonate or by ammonium sulfate precipitation failed to improve visualization and identification of proteins. The 2D gels of carbonate-washed membranes were largely devoid of proteins (only four proteins were identified), whereas 2DE of carbonate-soluble proteins failed to reveal proteins not found on gels of whole granule proteins (data not shown). Likewise fractionation of azurophil granule proteins by ammonium sulfate precipitation failed to improve the number of proteins visualized by 2DE (data not shown). Thus, only eight proteins from azurophil granules were identified by 2DE (Table I).

For all granule subsets, the protein fraction precipitated by 100% ammonium sulfate contained primarily luminal proteins. Only two proteins remained in solution after precipitation of specific and gelatinase granule proteins with 100% ammonium sulfate, MRP-14 and MRP-8, whereas the corresponding sample from azurophil granules was devoid of protein (data not shown). To identify any proteins that failed to dissolve in the TX-100-containing buffer and thus could not be detected in ammonium sulfate precipitated fractions, TX-100-insoluble pellets were also subjected to SDS-PAGE. Only actin, vimentin, CD11b, lactoferrin, and gelatinase were detected in pellets from specific and gelatinase granules (data not shown), whereas myeloperoxidase, cathepsin G, and lysozyme were found in the TX-100-insoluble fraction of azurophil granules. A total of 87 proteins were identified from granule subsets by the three 2DE approaches of which 56 were cytoskeletal or luminal proteins. Membrane proteins, except for CD11b and CD18, were not visualized by 2DE.

2DE-based approaches are biased against membrane proteins, low abundance proteins, and proteins at the extremes of isoelectric point and molecular mass. To address these issues, we used an approach that couples 2D HPLC with ESI-MS/MS analysis (22, 24). This high sensitivity mass spectrometry-based approach that allows direct analysis of complex protein mixtures was applied to granule membranes following removal of luminal and cytoskeletal proteins with sodium carbonate. Only proteins present in at least two of three experiments or proteins also identified by 2DE-MALDI MS were considered as valid granule proteins. By this criteria a total of 247 proteins were identified from all granule subsets of which 48 were also identified by 2DE. Table II lists identified proteins by granule subset, method of identification, and functional classification of the protein. A total of 86 proteins were identified only from gelatinase granules, 28 proteins were identified from only specific granules, and 26 proteins were identified from only azurophil granules. A number of proteins were identified on multiple granule subsets, including 79 proteins from gelatinase and specific granules, five proteins from specific and azurophil granules, and 62 proteins from all three granule subsets. The peptide sequences detected by 2D HPLC ESI-MS/MS can be found in Supplemental Tables 1 and 2. Supplemental Table 1 lists the proteins identified at least twice in three experiments and the sequences of corresponding peptides. Proteins identified once in a given granule subset by 2D HPLC ESI-MS/MS and identified by either 2DE or at least twice by 2D HPLC ESI-MS/MS in another granule subset are listed in Supplemental Table 2. Sequences of corresponding peptides and the percent coverage of identified proteins are also shown.


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TABLE II All proteins identified on neutrophil granules by 2DE MALDI-TOF MS and SCX-RP ESI-MS/MS

A total of 286 proteins were identified, and proteins were catalogued by function based on a literature search for each protein in the PubMed database. The gene and protein designation, method of identification, and granule subset on which the protein was found are listed. To be listed as a component of the granule subset, a protein was either identified from a granule subset by MALDI-MS analysis of 2D gels, detected by at least two of three 2D HPLC ESI-MS/MS experiments, or detected by 2D HPLC ESI-MS/MS experiment once in one granule subset and additionally detected by 2DE MALDI-MS or at least twice by 2D HPLC ESI-MS/MS in a different granule subset (indicated by an asterisk near the method designation in "Granule typeMETHOD" column). In the method designation, LCMS is an abbreviation for 2D HPLC ESI-MS/MS, and 2D is an abbreviation for 2DE MALDI-TOF MS. SOD, superoxide dismutase; hnRNP, heterogeneous nuclear ribonucleoprotein; GE, gelatinase; SG, specific granule; AG, azurophil granule; ICAM, intracellular adhesion molecule; CEACAM, carcinoembryonic antigen-related cell adhesion molecule; ADAM, a disintegrin and metalloprotease; LSP1, lymphocyte-specific protein 1; SNAP, synaptosomal-associated protein; ARP, actin-related protein; VAMP, vesicle associated membrane protein; Grp, glucose-regulated protein; Hsp, heat shock protein.

 
To eliminate proteins present as contaminants of granule subsets, proteins that were detected by only one of the three 2D HPLC ESI-MS/MS experiments were classified as rejected proteins. Supplemental Table 3 lists the rejected proteins for each granule subset. A total of 344 proteins were rejected from gelatinase granules, 211 were rejected from specific granules, and 230 were rejected from azurophil granules. Most rejected proteins were of ribosomal, nuclear, and mitochondrial origin or were hypothetical proteins, suggesting that the granule isolation method resulted in contamination with small amounts of other subcellular organelles. That some contamination did occur is supported by identification of several proteins listed in Table II. The benzodiazepine receptor was described previously to be present on mitochondria (29). Lamin B receptor and Sad1 are of nuclear origin (30, 31), suggesting that membranes from these structures contaminated granule preparations. The presence of signaling proteins (G protein-coupled receptor kinase 6; p21-activated kinase 5; small GTPases of the Ras, Rab, and Rho family; and heterotrimeric G protein subunits) and membrane trafficking proteins (syntaxin 2, syntaxin 3A, syntaxin 8, syntaxin 10, and syntaxin 11) in the list of rejected proteins suggests the exclusion criteria eliminated some proteins that are likely components of the granule proteome. Functional classification of proteins was performed by a literature search for each protein in the PubMed database.

Receptors and Cytoskeletal Membrane Anchors—
This group included transmembrane proteins involved in adhesion, transmigration, cellular activation, and cytoskeletal anchoring to the membrane, including components of lipid rafts. Of the 40 proteins, 12 were identified only from gelatinase granules, 19 were found on both specific and gelatinase granules, six were found on all three granule types, and three were found only on azurophil granules. Proteins unique to specific granules were not found in this group. Formyl peptide receptors, complement component receptor 1 (CR1), CD11b, CD18, SCAMPs, Stomatin, CD63, and LAMPs have been identified previously as components of neutrophil granules (1, 2, 32).

Channels and Transporters—
Proteins that function in facilitating the movement of solutes across lipid bilayers were included in this category. Thirteen proteins were identified only on gelatinase granules, 10 were from gelatinase and specific granules, two were from all three granule subtypes, and two were only from azurophil granules. These proteins included proton pumps and transporters for metal ions, glucose, adenine nucleotides, amines, and steroids. Eleven of these proteins were described previously to be of mitochondrial origin.

GTPases—
This group included both monomeric and heterotrimeric GTPases and two GTPase-activating proteins. Of the total of 21 identified proteins, 10 were found on gelatinase granules only, one was only from specific granules, two were from specific and gelatinase granules, and eight were from all three granule subsets. Monomeric GTPases of the Rab family and Cdc42 have been shown to be involved in granule exocytosis in other cell types (3335). Of note, Rab27A was shown previously to associate with many types of granules and play a crucial role in exocytosis by modulating granule binding with cytoskeleton and with proteins that control membrane fusion (36). Rab27A was a component of all three granule types. To our knowledge this is the first report of the presence of Rab27A in neutrophils. Cdc42 was also found on all three granule subsets. This Rho family GTPase stimulates exocytosis by activating the inositol 1,4,5-trisphosphate and calcium second messenger signaling pathway (35).

Structural Proteins and Adaptors—
We included cytoskeletal proteins and actin-binding proteins in this group. Eleven proteins were identified only on gelatinase granules, 13 were from gelatinase and specific granules, four were from specific granules only, 10 were from all three granule types, and two were from only azurophil granules. The presence of actin, tubulin, and vimentin on all three granule subtypes is consistent with a role for these cytoskeletal structures in neutrophil exocytosis (1, 2, 37, 38). Visual inspection of 2D gels suggested that greater amounts of cytoskeletal proteins were associated with gelatinase granules than specific granules, which in turn had more than azurophil granules. To confirm this observation, Western blotting of whole granules for actin was performed following normalization for membrane content using a membrane-binding fluorescent dye (Fig. 5). The amount of actin associated with gelatinase granules was dramatically greater than that associated with specific granules. A minimal amount of actin was associated with azurophil granules.



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FIG. 5. Assessment of actin content on neutrophil granules by Western blotting. Membrane lipid content was quantitated in neutrophil granules by TMA-DPH lipophilic dye, and granule proteins were loaded such that amounts of granule membranes loaded were equal among different granule subsets. Proteins were transferred to nitrocellulose and probed for actin with an anti-actin antibody.

 
Kinases and Phosphatases—
This group included tyrosine kinases, tyrosine phosphatases, and one kinase scaffolding protein, MEK1-binding protein. Tyrosine kinases were identified previously on neutrophil granules, and inhibition of tyrosine kinase activity prevents neutrophil degranulation (3942). To our knowledge, this is the first report of a tyrosine phosphatase association with neutrophil granules. Tyrosine phosphatases are localized to granules in other cells, and they have been shown to participate in signaling leading to exocytosis (4346).

Luminal and Host Defense Proteins—
This group included proteins localized to granule lumens and luminal and membrane proteins that participate in host defense, such as the membrane proteases neprilysin and leukolysin. Four proteins were identified on gelatinase granules only, 13 proteins were from gelatinase and specific granules, 11 were proteins only from specific granules, 18 were from all three granule subsets, three were from specific and azurophil granules, and 13 were from azurophil granules only. All of the luminal proteins in this list are annotated on the NCBI database as proteins destined either to lysosomal or to secretory compartments. Proteins of lysosomal origin were found previously in azurophil granules in agreement with the hypothesis that azurophil granules are lysosome-related organelles (1, 2). Among the identified proteins in this group was calreticulin, a component of the endoplasmic reticulum (47). Calreticulin may also be a bona fide granule protein, however, as it acts as a chaperone for neutrophil granule luminal proteins (48) and is present on the extracellular surface of the neutrophil plasma membrane (49). Calreticulin was identified as a cytolytic component of T lymphocyte granules (50).

Membrane Traffic and Fusion—
In this group four proteins were found on gelatinase granules only, one was from gelatinase and specific granules, two were from specific granules alone, four were from all three granule types, and two were from azurophil granules only. Hunc18b (Munc18-2) and Unc-13 homolog 3 (Munc-13) have not been previously identified in neutrophils. These proteins are members of a family of proteins that bind to and modulate the activity of membrane fusion proteins (51, 52). A hypothetical protein with a C2 domain, which is known to be involved in membrane fusion events (53), was identified on gelatinase granules. Syntaxin 7 and VAMP 8 were expressed on all three granule subsets. These two membrane fusion proteins have not been previously identified from neutrophil granules.

Redox Proteins—
Proteins involved in redox reactions, including a transmembrane protein related to thioredoxin and the components of the neutrophil NADPH oxidase, p22-phox and gp91-phox, were included in this group. Ten proteins were found only on gelatinase granules, one was from gelatinase and specific granules, two were from specific granules alone, and three were from all three granules. Components of the NADPH oxidase were found on all three granule subsets.

Miscellaneous Proteins—
This group included proteins not classified into the other groups or for which no function has been described. Twenty-one proteins were identified on gelatinase granules, 16 were from gelatinase and specific granules, six were from specific granules only, 10 were from all three granule types, two were from specific and azurophil granules, and four were from azurophil granules only. Among the proteins found were chaperones such as PDI and Hsp70 and 11 hypothetical proteins. Hemoglobin was also found on gelatinase and specific granules, suggesting that hemoglobin from lysed red blood cells binds to phospholipid membranes. Hemoglobin was completely removed from granule membranes by sodium carbonate treatment.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using two approaches, 2DE followed by MALDI MS and 2D HPLC ESI-MS/MS, 286 granule and granule-associated proteins from human neutrophils were identified. Only 87 proteins were identified by 2DE and MALDI MS of which 56 were abundant structural or luminal proteins. The only transmembrane-spanning proteins identified after 2DE were the integrins CD11b and CD18, both of which have a single transmembrane domain. The limited protein identification using intact granules was marginally enhanced by fractionation of granule proteins using sodium carbonate treatment or ammonium sulfate precipitation. These results emphasize the recognized limitations to 2DE-based proteomics, namely, the bias against low abundance proteins, transmembrane proteins, and proteins at the extremes of isoelectric point and molecular mass (54, 55). Despite these limitations, 39 proteins were identified only by the 2DE-based approach.

The 2DE analysis of whole granules revealed that the quality of protein separation paralleled the density of the lumen matrix for each granule subset (1, 2, 17, 25). This is likely due to the presence of large amounts of basic proteins in the granule lumens that interfere with IEF and render the less abundant membrane-associated proteins unable to focus. This observation led to attempts to fractionate granule proteins with alkaline sodium carbonate or precipitation with various concentrations of ammonium sulfate. The sodium carbonate method fractionated granule proteins largely into luminal and membrane fractions. This method resulted in effective 2DE separation of specific granule proteins possibly because these granules contain large amounts of moderately cationic luminal proteins that can be dissociated from the luminal matrix and granule membranes. The less dense granule matrix of gelatinase granules was associated with adequate protein resolution by 2D gels, resulting in minimal improvement after sodium carbonate extraction of luminal proteins. Sodium carbonate extraction did not improve separation and identification of proteins from azurophil granules, which contain a highly packed matrix of acid mucopolysaccharide and highly cationic myeloperoxidase (56). Sodium carbonate treatment of gelatinase, but not specific granules, also resulted in the significant loss of low molecular weight proteins compared with 2DE of whole granules (compare Fig. 2A with Fig. 3, A and B).

The failure of sodium carbonate extraction to improve protein identification of gelatinase or azurophil granules prompted us to seek an alternative method of protein fractionation based on a different physical characteristic. Ammonium sulfate precipitation fractionates proteins according to their solubility in concentrated ammonium sulfate solutions. For neutrophil granules, this method separated cytoskeletal and cytoskeleton-binding proteins from more hydrophilic luminal proteins. This fractionation method enhanced protein separation and identification of gelatinase granule proteins (Fig. 3, C and D). Fractionation of specific and azurophil granule proteins by ammonium sulfate precipitation did not improve protein separation or identification. This was likely due to the fact that most of the protein content in these granules was of luminal origin and, therefore, hydrophilic. Such proteins precipitate at high concentrations of ammonium sulfate, and therefore they precipitated in the 100% ammonium sulfate solution. More hydrophobic proteins, which were mostly cytoskeletal and were less abundant, precipitated in the 20% ammonium sulfate solution. The use of other concentrations of ammonium sulfate did not result in improved fractionation of granule proteins (data not shown). Thus, sodium carbonate treatment was more effective for identification of specific granule proteins, whereas ammonium sulfate precipitation was more effective for identification of gelatinase granule proteins. Neither approach improved protein identification from azurophil granules. These results indicate that optimal protein extraction and separation of granule proteins by 2DE is highly dependent on the properties of the granule matrix and luminal proteins.

To overcome the limitations of 2DE-based protein identification, granule proteins were also identified by strong cation exchange reverse phase two-dimensional chromatography combined with ESI-MS/MS, commonly referred to as direct analysis of large protein complexes (DALPC) or multidimensional protein identification technology -spanning and membrane-associated proteins, sodium carbonate extraction was used to remove luminal and cytoskeletal proteins (18, 2628, 58). As shown by the comparison of Fig. 2 with Figs. 3 and 4, the use of sodium carbonate significantly reduced high abundance luminal proteins, whereas the reduction in cytoskeleton was modest. 2D HPLC ESI-MS/MS identified 247 proteins from the three granule subsets.

The distribution of proteins among the granule subsets indicates that approximately half were present on more than one granule subset. The presence of proteins in more than one granule subset could be due to neutrophil granule biogenesis or to cross-contamination among the granule subsets following separation by Percoll gradient centrifugation. Granule biogenesis occurs during myeloid cell maturation with different granules forming at different stages of differentiation. The protein content of these granules is determined by the timing of protein synthesis relative to formation of different granule subsets, not by selective targeting of proteins to different granules (17). The relative purity of granule subset preparations was addressed to determine the quality of the proteome for each granule. The distribution of markers for each granule subset showed granule separation similar to that reported by other groups (17, 25). Western blot analysis for CD66b, a marker for specific granules, showed that gelatinase and azurophil granules were not contaminated by specific granules. The presence of gelatinase in the specific granule preparation is consistent with a previous study showing that 60% of myeloperoxidase-negative granules contain gelatinase in addition to a marker of specific granules, lactoferrin (1, 2). The presence of myeloperoxidase in specific and gelatinase granules may be due to the extremely basic nature of this protein, which allows myeloperoxidase released from azurophil granules to bind to phospholipid membranes of other granules. Based on current separation techniques and known granule markers, however, minimal cross-contamination among the granule subsets cannot be excluded.

We attempted to establish a valid list of proteins for each granule type by rejecting proteins that were identified in only one of three 2D HPLC ESI-MS/MS experiments. A total of over 500 proteins, including nuclear, mitochondrial, ribosomal, and cytosolic proteins, were rejected using this exclusion criterion. A few contaminating proteins may have still been present in granule proteomes as several mitochondrial membrane transporters were identified in more than one granule preparation, especially in the lighter gelatinase granule fraction. This is likely due to the fact that gelatinase granules sediment at a density of 1.08 g/ml in Percoll (17, 25), whereas mitochondria sediment at a density of 1.05 g/ml (59). Histones were also identified in all three granule subsets. Their presence may reflect nuclear contamination from the reported breakage of 16% of nuclei during nitrogen cavitation of neutrophils (17, 25) and subsequent nonspecific binding to granule membranes. Histones recently were described to co-localize with granule enzymes in neutrophil extracellular traps (60). Subcellular fractionation showed that murine macrophage granules contained histones (61). Histones were also secreted by amnion cells (62). Thus, histones may be components of neutrophil granules. The list of rejected proteins contained several proteins, including syntaxins and synaptobrevin-like proteins, that regulate granule membrane fusion. Although none of these proteins have been identified previously on neutrophil granules, these results suggest the possibility that some granule proteins may have been excluded by the criteria used in this study. The presence of false positive and false negative results indicates that defining subcellular organelle proteomes using highly sensitive mass spectrometry-based techniques is limited by the ability to purify these organelles. Ultimately confirmation that a specific protein is present in an organelle will require histologic studies.

Analysis of the distribution of proteins among the granule subsets revealed differences among the subsets that suggest functional heterogeneity. The total number of proteins and the number of proteins in each functional classification, except for luminal proteins, increased from azurophil to specific to gelatinase granules. The number of luminal proteins identified was greatest in specific granules (45 proteins), whereas azurophil (34) and gelatinase (35) granules contained similar number of proteins. Gelatinase granules are the most easily mobilized granules in neutrophils, and, therefore, these granules will undergo exocytosis at an earlier stage of neutrophil activation than specific or azurophil granules. Gelatinase granule membranes contain a large number of membrane receptors and adhesion molecules and are associated with the largest amount of actin cytoskeleton and cytoskeletal regulatory proteins. These results support a role for gelatinase granules in enhancing the plasma membrane expression of molecules necessary for neutrophil adherence to and migration through inflamed vascular endothelium and for subsequent chemotaxis. On the other hand, azurophil granules primarily fuse with phagosomes, while exocytosis is negligible. These granules contain the largest number of luminal bactericidal proteins, while there is a paucity of membrane, cytoskeletal, and GTP-binding proteins. The greater complexity of the specific granule lumen and the presence of a significant number of transmembrane and membrane-associated proteins suggest that these granules represent a transitional phase that can contribute to neutrophil activation through exocytosis or provide bactericidal proteins to phagosomes. Our results also suggest a differential role of the actin cytoskeleton in regulation of granule exocytosis. Gelatinase granules, the most easily mobilizable granule subset, are associated with the largest amount of actin cytoskeleton. On the other hand, azurophil granules are associated with very little actin, and they fail to exhibit exocytosis unless the subplasma membrane actin cytoskeleton is disrupted (1, 2). Once again, specific granules represent a transition between these two extremes. Previous reports have emphasized the role of the subplasma membrane actin cytoskeleton as a barrier to granule exocytosis (63). The present study suggests that the actin cytoskeleton associated with granules also plays an active role in mediating exocytosis.

To our knowledge, this is the first proteomic study of neutrophil granules. The importance of regulated exocytosis in determining the activation state of neutrophils and the use of regulated exocytosis by a large number of other cells indicate the value of defining granule proteomes. The methodology described herein provides an approach to defining granule proteomes. In our experience, 2DE identified and revealed relative distribution of more abundant proteins in granules, whereas 2D HPLC ESI-MS/MS identified less abundant proteins and hydrophobic proteins not amenable to detection by 2DE. The sensitivity of this latter approach demands that granules be isolated to a high degree of purity and/or experimental approaches be used to eliminate false positive protein identification. Our data show that neutrophil granules are complex organelles. Analysis of the more than 200 proteins associated with neutrophil granules will allow a better understanding of the contribution of each of the granule subsets to neutrophil biology.


   FOOTNOTES
 
Received, May 17, 2005

Published, MCP Papers in Press, June 28, 2005, DOI 10.1074/mcp.M500143-MCP200

1 The abbreviations used are: 2DE, two-dimensional gel electrophoresis; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate; CR1, complement component receptor 1; MEK1, extracellular signal-regulated kinase (ERK) activator kinase 1; Hsp70, heat shock protein 70; MPO, myeloperoxidase; NGAL, neutrophil granule-associated lipocalin; PDI, protein-disulfide isomerase; 2D, two-dimensional; TX-100, Triton X-100; PIPES, 1,4-piperazinediethanesulfonic acid; MRP, myeloid-related protein; SCAMP, secretory carrier membrane protein; LAMP, lysosome-associated membrane protein; VAMP, vesicle-associated membrane protein. Back

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

** To whom correspondence should be addressed: Molecular Signaling Group, Kidney Disease Program, Donald E. Baxter Research Bldg., 570 S. Preston St., University of Louisville, Louisville, KY 40202. Tel.: 502-852-0014; Fax: 502-852-4384; E-mail: k.mcleish{at}louisville.edu


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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