Identification of Human Olfactory Cleft Mucus Proteins Using

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Identification of Human Olfactory Cleft Mucus Proteins Using Proteomic Analysis He´ le` ne De´ bat,*,†,O Corinne Eloit,‡,⊥ Florence Blon,† Benoıˆt Sarazin,§ Ce´ line Henry,| Jean-Claude Huet,† Didier Trotier,‡ and Jean-Claude Pernollet† Biochimie de l’Olfaction et de la GustationsUMR 1197sNeurobiologie de l’Olfaction et de la Prise Alimentaire (NOPA), INRA, Jouy-en-Josas, France, Neurobiologie SensoriellesUMR 1197sNOPA, INRA, Jouy-en-Josas, France, Proteomics Solutions, Saint-Marcel, France, Plateau d’Analyse par Se´quenc¸ age et Spectrome´trie de massesBiochimie Bacte´rienne, INRA, Jouy-en-Josas, France, ENT Department, Hoˆpital Lariboisie`re, Paris, France, Medical Center Institut Pasteur, Paris, France, and Universite´ Versailles St-Quentin en Yvelines, Versailles, France Received December 8, 2006

In humans, the olfactory epithelium is located in two narrow passages, the olfactory clefts, at the upper part of the nasal cavities. The olfactory epithelium is covered by a mucus layer which is essential for the function of the olfactory neurons that are directly connected with the brain through the cribriform plate. This anatomical weakness of the brain protection may be the source of infection. Little is known about the composition of this mucus in humans. Previous proteomic analyses have been performed on washes of the entire nasal cavities and therefore might better correspond to the mucus over the respiratory epithelium than to the mucus covering the olfactory epithelium. In the present study, we sampled the olfactory mucus directly from the clefts of 16 healthy adult volunteers, and 83 proteins were identified in the samples using two-dimensional gel electrophoresis, MALDI-TOF, RPLC, and Edman sequencing. Forty-three proteins were not previously observed either in nasal mucus sampled through washings, saliva, tear, or cerebrospinal fluid. In Accordance with the data in the protein databases, the most abundant proteins are secreted, whereas some others correspond to intracellular proteins covering a large range of functions: anti-inflammatory, antimicrobial, protease inhibition, antioxidant, transport, transcription, transduction, cytoskeletal, regulation, binding, and metabolism of odorant molecules. This study clearly demonstrates the complexity of the mucus covering the human olfactory epithelium, which might comprise potential markers for characterizing pathophysiological states. Keywords: Anti-inflammatory proteins • Human proteome • Odorant-binding protein • MALDI-TOF • Twodimensional gel electrophoresis

The first step in the olfactory perception is the activation by odorants of sensory neurons in the olfactory epithelium (OE). In humans, this epithelium is located at the level of two narrow passages, the olfactory clefts, at the upper part of each nasal cavity. In humans, a specific obstructive inflammation in the olfactory clefts, with no sign of obstruction or inflammation in the rest of the nasal cavity or sinuses, leads to a deterioration of olfaction similar to the absence of olfactory bulbs.1,2 Biopsies

reveal the presence of the OE at this level.3-5 The epithelium contains neurons which detect the presence of odorant molecules and transmit the information through the cribriform plate to the olfactory bulbs. Electrophysiological recordings indicate that the epithelium may extend, in some individuals, to the anterior part of the clefts.6,7 Before reaching the ciliary membrane of the olfactory neurons, odorant molecules must diffuse through the mucus covering the OE. Bowman’s glands and supporting cells in OE contribute to the secretion of the mucus.8

* Corresponding author: He´le`ne De´bat, Biochimie de l’Olfaction et de la GustationsUMR 1197sNOPAsBaˆt. 526sINRA, Domaine de Vilvert - F-78352 Jouy-en-Josas CEDEX, France. E-mail: [email protected]. Fax: +33134652765. † Biochimie de l’Olfaction et de la GustationsUMR 1197sNeurobiologie de l’Olfaction et de la Prise Alimentaire (NOPA), INRA. ‡ Neurobiologie SensoriellesUMR 1197sNOPA, INRA. § Proteomics Solutions. | Plateau d’Analyse par Se´quenc¸ age et Spectrome´trie de massesBiochimie Bacte´rienne, INRA. ⊥ ENT Department, Hoˆpital Lariboisie`re; Medical Center Institut Pasteur. O Universite´ Versailles St-Quentin en Yvelines.

Little is known about the composition and the properties of this mucus at the level of the olfactory clefts. The proximity of the brain can be a source of infection and therefore requires thorough knowledge. Previous studies examined the protein composition of washes of the nasal cavities.9-21 However, these samples most probably correspond more to the mucus covering the nonolfactory regions of the cavities (septum and lower and middle turbinates) rather than the olfactory clefts which are narrow, difficult to reach, and correspond to a small surface area.

Introduction

10.1021/pr0606575 CCC: $37.00

 2007 American Chemical Society

Journal of Proteome Research 2007, 6, 1985-1996

1985

Published on Web 03/24/2007

research articles We recently developed a method to sample the mucus directly from the clefts using a thin catheter inserted into the cleft.9 We found that the mucus in the clefts displayed significantly different reverse-phase liquid chromatography (RPLC) elution diagram profiles when compared to the mucus sampled at the level of the septum or the middle turbinate. Particularly, fragments of odorant-binding proteins (OBPs) were detected in the olfactory cleft mucus but not in the mucus covering other areas of the cavity.9 OBPs, which are soluble proteins presenting an internal hydrophobic pocket able to carry odorant lipophilic molecules,22,23 are considered to be important for olfactory processes. This study has clearly demonstrated that key elements for olfaction are specifically located in the mucus covering the OE in humans. Another aspect of the role of the olfactory mucus is the possible modification of odorant molecules. These modifications are conceivable as various enzymes have already been observed in the olfactory mucus of other species, including odorant degradating enzymes.24 Finally, the olfactory mucus should present protective properties and act as a barrier against infections. The sensory neurons are indeed in direct contact with the brain through the sensory axons which cross the cribriform plate to make synapses in the olfactory bulb, and it has already been demonstrated, in rodents, that this anatomical weakness of the brain protection may be the source of brain infection. For example, neuroadapted viruses actively proliferate in the receptor cells and migrate to the olfactory bulb in mice,25 and herpes virus EHV-9 causes fulminant encephalitis in dog through infection of the peripheral olfactory system.26 In the present study, we therefore considered of prime interest to investigate the protein content of the mucus directly sampled at the level of the clefts. We used a proteomic approach to determine the nature of these proteins, using twodimensional gel electrophoresis (2-DE), MALDI-TOF, RPLC, and Edman sequencing, in order to clarify their role in the physiology of the olfactory clefts.

Materials and Methods Olfactory Cleft Samples. Samples were obtained using a sterile polyethylene catheter (Merck-Boiron, France, 0.7 mm internal diameter, 1.09 mm external diameter) connected to a vacuum pump. Sampling was performed on 16 healthy adult volunteers (7 males, 21-58 years old; and 9 females, 21-56 years old) lying supine in a quiet room by an ENT medical surgeon (Dr. Corinne Eloit) at Lariboisie`re Hospital (Paris, France). Although painless without inherent risk, the sampling procedure was performed according to the Declaration of Helsinki recommendations (DHEW Publication, NIH 86-23) for nontherapeutical clinical research. The subjects were fully informed about the nature, the purpose, and the potential risks and gave their free consent. Smokers and subjects with inflammatory nasal mucosa were discarded from this study. Under endoscopical control (Storz Foreward Endoscope 0 or 30°, 4-mm external diameter supplied with a cold light), a slight anaesthesia of the lower part of the nasal cavity was performed. A cotton swab soaked with xylocaine in 5% naphazoline (AstraZeneca) was introduced in the nasal fossae covering the inferior turbinates on both sites. Ten minutes later, swabs were removed and the catheter was gently introduced into the nasal cavity up to the uppermost part of the nasal cavity, reaching the olfactory cleft, which is hardly directly visible using an endoscope, except in its most anterior part, 1986

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De´ bat et al.

above the middle turbinate.27,28 Gentle suction was applied to the catheter. Individual sample volumes varied between 1 and 4 µL of mucus, depending on subjects. Mucus was pooled from 10-12 healthy subjects at each experiment. Sixteen healthy different subjects took part in the sampling. Samples were immediately frozen in liquid nitrogen and kept at -80 °C until use. All manipulations of the samples were performed by people wearing gloves and caps to prevent contamination. Reverse-Phase High-Performance Liquid Chromatography. Approximately 4 µL of sample was diluted to 20 µL by solvent A (0.1% v/v formic acid and 4 mM ammonium acetate in water). RPLC was run with an Applied Biosystems device (pump 140D and UV detector 785 with U-shaped fused-silica tubing, 7-mm path length) on a C4 LC-Packings capillary column (0.3 × 150 mm, 300 Å) at controlled temperature (40 °C). The gradient was made by mixing solvent A with solvent B (90% acetonitrile (ACN), 0.1% v/v formic acid, and 4 mM ammonium acetate in water) at a flow rate of 4 µL/min. The ACN gradient began at 4.5% and increased linearly to 85.5% in 90 min. The flow rate was monitored by photometry at 215 nm, and fractions were manually collected. Edman Sequencing. N-terminal amino acid sequence analyses were performed by automated Edman chemistry using a Procise 494 HT protein sequencer (Perking-Elmer) with methods and reagents of the manufacturer. 2-D Gel Electrophoresis. Sample proteins were solubilized while they were pushed in the catheter using a needle and a syringe containing 600 µL final volume of 9 M urea (m/v), 4% CHAPS (m/v), 0.6% ampholytes, and 20 mM DTT (m/v) and pooled. Between 200 and 250 ng of protein was used to run 2-DE. The first dimension of the 2-DE was performed in a horizontal apparatus (Protean IEF Cell, Bio-Rad, Hercules, CA) with pH 3-10 strips for 115 000 Vh. IPG strips were equilibrated and fixed on top of 1.5-mm-thick slab gels consisting of 16% polyacrylamide (Duracryl, Genomic Solutions). The gels were assayed on a vertical electrophoresis unit (Dodecacell, Bio-Rad) at a constant 200 V for the second dimension. When migrations were completed, gels were stained for 5 days with Coomassie Colloidal blue and then destained in 1% acetic acid (v/v) for at least 24 h. Image Analysis. Gels were scanned at a resolution of 300 dpi with a Powerlook II scanner (UMAX); pictures were stored as TIFF format and further were processed with the Melanie 4 computer system (Genebio, Geneva). Protein spots were outlined automatically using Melanie 4 software. Spot detection was checked manually. Matching of 2-D gel patterns was performed by selecting approximately 10 spots clearly identified and observed in every gel. Quantitative and qualitative information of the spots were then reported (spot ID, relative % optical density, relative volume, relative area, estimated MW, and pI). Tryptic Digestion. The excised gel plugs (∼ 3.5 mm3) were washed in 50% ACN and 50 mM NH4CO3 (v/v) in Eppendorf tubes. After gel-drying in a Speed Vac Concentrator (Speed Vac Plus, SC100A, Savant) for 30 min, the digestion was performed in 25 µL of 50 mM ammonium bicarbonate, pH 8.0, with 0.5 µg of modified trypsin (Promega, sequencing grade) for 6 h in a thermomixer (Eppendorf) at 37 °C with vortexing at 500 rpm. The supernatant of tryptic digest was put in Eppendorf tubes, and peptides still retained in pieces of gel were extracted two times in 50% ACN with 5% TFA (v/v). All liquids were pooled and dried in a Speed Vac Concentrator. Resulting peptides were directly resuspended in 10 µL of TFA 0.3%.

Identification of Human Olfactory Cleft Mucus Proteins

research articles performed on each 2-DE and, in order to examine the reproducibility of samples, 2-DE protein profiles were compared to each others using Melanie 4 software. Protein spots were excised and digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF for protein identification. By the use of this strategy, 126 observed spots were excised and 111 were identified. These 126 spots observed were found in at least 10 2-DE among 48 which were performed in this study. The 2-DE shown in Figure 1 is chosen as the most representative among all the electrophoregrams which present weak interand intravariations between all samples. Weak variations were observed in low protein molecular weight region that often comprises protein fragments.

Results and Discussion

Database search results were only accepted if the score reported by MS-FIT was higher than 1700, provided that the percentage of coverage was over 15% and, whenever possible, attributions were validated with MW/pI values (see Supporting Information data table). We checked manually that all the major peaks of the mass spectra matched to the peptides of protein identified by the MS-FIT. In these conditions, 75 distinct proteins were identified, which are listed in Table 1 and numbered according to Figure 1. The discrepancy of this value with the number of observed spots can be attributed to the presence of isoforms, post-translational modifications, and/ or protein fragmentation, which occurred in different spots identified as a single accession number. Only the most frequent protein fragments are ticked on the 2-DE in Figure 1. The protein identification frequency is also listed in Table 1 where one can observe that the lowest frequencies, between 50 and 60%, correspond to proteins whose quantities are low in 2-DE and, thus, could not systematically be identified by the MALDITOF technique. The major part (85%) of the observed proteins is found between 20 and 80 kDa MW and between pH 5 and 8. According to Table 1 numbering, the most abundant wellknown proteins we found in OCM were serum albumin (no. 66), serotransferrin (no. 65), alpha-1-antitrypsin (no. 6), actins (nos. 1 and 2), squamous cell carcinoma antigen (no. 67), palate lung nasal epithelial clone (PLUNC) protein (no. 62), apolipoprotein A-1 (no. 14), and glutathione S-transferase P (no. 33). Among these abundant proteins, serum albumin (no. 66) is largely the most abundant and constitutes a horizontal streaking (pI 6-7) around 69 kDa. This indicates polymorphic posttranscriptional modifications (likely variable phosphorylation). Similar phenomena were also observed in all 2-DE diagrams for serotransferrin (no. 65) and alpha-1-antitrypsin (no. 6), although not clearly visible in Figure 1. Aside from posttranslational side chain modifications, we found a vast assortment of albumin degradation products. Since albumins are known to be the most abundant protein in human serum,34 it was therefore necessary to zoom in the pH focusing between 5 and 8 and polyacrylamide concentration of 9%. Seventy-six spots were excised, and 56 proteins were identified without revealing any new protein (data not shown). Nevertheless, some proteins were detected in spite of their low molecular weight (nos. 20, 21, 39, 40, and 52) and/or basic pI (nos. 32, 39, 52, and 54) among which lysozyme-C (no. 52) was found to be very abundant.

Olfactory Cleft Mucus (OCM) Proteome Description. The OCM proteome was first studied using the combination of 2-DE and MALDI-TOF analysis. After protein denaturation, sample was separated by 2-DE (Figure 1). Two to four 2-DE gels were performed with each mucus sample depending on the total mucus quantity collected. Qualitative analyses were separately

As a complementary approach, we have analyzed some OCM samples using a RPLC and Edman sequencing as previously described by Briand et al.9 This approach allowed us to complement the OCM protein cartography by identifying 8 additional proteins not observed with the 2-DE-coupled MALDITOF approach. Proteins identified using the RPLC procedure

Figure 1. 2-DE pattern of olfactory cleft mucus proteins. Proteins were separated by analytical 2-DE and detected by Coomassie blue staining. Numbers refer to identified proteins in Table 1. Scanned gel presented as an unmanipulated image, except for overall contrast and light, modified using Melanie 4 software (Genebio).

Mass Spectrometry. One microliter of sample was mixed on the stainless steel MALDI plate with 1 µL of CHCA (R-cyano4-hydroxycinnamic acid, Sigma Aldrich) at 4 mg‚mL-1 in ACN/ TFA 0.3% (50:50; v/v) and dried at room temperature. Mass spectra were acquired on a Voyager DE-STR+ time-of-flight mass spectrometer (Applied Biosystems) equipped with a nitrogen laser emitting at 337 nm. Spectra were recorded in positive reflector mode with 20 kV as accelerating voltage, a delayed extraction time of 130 ns, and a 62% grid voltage. In case of high background noise, the CHCA matrix was replaced with DHB matrix (2,5 dihydroxybenzoic acid, Sigma Aldrich) at 10 mg.mL-1 in ACN/TFA 0.3% (50:50; v/v). Ammonium phosphate (10 mM final concentration) was added to peptides of interest whose peaks were overlapped by matrix clusters peaks of CHCA. Spectra were obtained in the mass range between 700 and 4000 Da and were calibrated using internal calibration with autolytic trypsin fragments characterized by (M + H)+ ) 842.509 and 2211.104 Da. Database Search. Proteins were identified using the protein sequence databases search program MS-FIT (http://prospector.ucsf.edu/prospector/4.0.7/html/msfit.htm) and the database Swiss-Prot2005.05.06. This peptide mass fingerprinting tool from the University of California at San Francisco aims at fitting a user’s mass spectrometry data to a protein sequence in an existing database in order to suggest the identity of the input protein. The following general search parameters were used: monoisotopic molecular masses, enzyme specificity of trypsin, and peptide tolerance of (0.04 Da. The search was restricted to the Homo sapiens database, and 35-165 masses were submitted. Protein description was established using Swiss-Prot,29 Pfam,30 UniProt,31 InterPro,32 and PROSITE33 database queries.

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Table 1. Proteins Identified in Human Olfactory Cleft Mucusa

no.

protein name

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Actin, cytoplasmic 1 (Beta-actin) Actin, cytoplasmic 2 (Gamma-actin) Alcohol dehydrogenase [NADP+] Aldehyde dehydrogenase 1A1 Aldehyde dehydrogenase, dimeric NADP-preferring Alpha-1-antitrypsin (precursor) Alpha enolase, lung specific Alpha-enolase (plasminogen-binding protein) Annexin A1 (lipocortin I) Annexin A2 (lipocortin II) Annexin A3 (lipocortin III) Annexin A7 (Synexin) Antileukoproteinase 1 Apolipoprotein A-I (precursor)* Apolipoprotein A-IV (precursor) ATP synthase beta chain, mitochondrial (precursor) Basic proline-rich peptide P-E Basic Salivary proline-rich protein 4 allele S Calcyphosine Calgranulin A* Calgranulin B Calreticulin (precursor) (calregulin) Carbonic anhydrase I Chloride intracellulaire channel protein 1 Creatine kinase, B chain Cystatin SN or salivary cystatin SA-1 Cytosol aminopeptidase Elongation factor Tu, mitochondrial (precursor) Epithelial membrane protein-2 Glucose-regulated protein 78 kDa (GRP 78) (precursor) Glutamate dehydrogenase 1, mitochondrial (GDH) (precursor) Glutathione S-transferase A1 Glutathione S-transferase P Glyceraldehyde-3-phosphate dehydrogenase Heat shock cognate 71 kDa protein (HSC 70) Heat shock protein beta-1 (HSP 27) Heat shock protein 60 kDa (precursor) (HSP 60) Heat shock 70 kDa protein 1 (HSP 70-1) Hemoglobin alpha chain* Hemoglobin beta chain* Hemopexin (precursor) Heterogenous nuclear ribonucleoprotein K Immunoglobulin J chain Isocitrate dehydrogenase (NADP) Keratin, type I cytoskeletal 18 Keratin, type I cytoskeletal 19 Keratin, type II cytoskeletal 8 Keratin, type II cytoskeletal 6D Keratin, type II cytoskeletal 7 (Sarcolectin) Lactotransferrin (precursor) (Lactoferrin) Lipid-binding-protein 4 (Sequence 3 from Patent WO0179269 MERCK PATENT GmbH) Lysozyme C (precursor)* Odorant-binding protein (hOBP)

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1988

Peroxiredoxin 1 Peroxiredoxin 2 Peroxiredoxin 5, mitochondrial (precursor) Peroxiredoxin 6 Phosphoglycerate mutase 1 Polymeric-immunoglobulin receptor (precursor)* Prohibitin Protein disulfide-isomerase A3 (precursor) Protein PLUNC (precursor) Pyruvate kinase, M1/M2 isozymes Selenium binding protein 1 Serotransferrin (precursor) (Transferrin, Siderophilin) Journal of Proteome Research • Vol. 6, No. 5, 2007

Swiss-Prot accession number

theorical MW (Da)/pI

P60709 P63261 P14550 P00352 P30838 P01009 Q05524 P06733 P04083 P07355 P12429 P20073 P03973 P02647 P06727 P06576

41.737/5.29 41.793/5.31 36.442/6.35 54.731/6.30 50.379/6.11 46.737/5.37 49.500/5.78 47.038/6.99 38.583/6.64 38.473/7.56 36.244/5.63 52.739/5.52 11.726/9.11 30.778/5.56 45.371/5.28 56.560/5.26

16 50% 17 55% 15 51% 19 48% 15 35% 20 48% 12 26% 20 55% 19 61% 26 59% 12 36% 17 32% RPLC, Edman sequencing 17 60% 12 37% 25 58%

a, c, d a a a a a, d d c a, c, d a

P02811 P10163 Q13938 P05109 P06702 P27797 P00915 O00299 P12277 P01037 P28838 P49411 P54851 P11021

6.024/11.74 25.108/10.42 20.967/4.74 10.835/6.51 13.242/5.71 48.142/4.29 28.739/6.63 26.792/5.09 42.644/5.34 16.362/6.82 52.640/6.29 49.542/7.26 19.199/7.55 72.333/5.07

RPLC, Edman sequencing RPLC, Edman sequencing 14 58% 8 49% 14 85% 11 31% 11 60% 15 71% 15 48% RPLC, Edman sequencing 27 58% 21 51% RPLC, Edman sequencing 21 39%

c

P00367

61.398/7.66

P08263 P09211 P04406 P11142 P04792 P10809 P08107 P69905 P68871 P02790 P61978 P01591 O75874 P05783 P08727 P05787 P48667 P08729 P02788

25.500/8.92 23.225/5.44 35.922/8.58 70.898/5.37 22.783/5.98 61.055/5.70 70.052/5.48 15.126/8.73 15.867/6.81 51.676/6.55 50.976/5.39 15.594/4.62 46.659/6.53 47.927/5.34 44.106/5.05 53.543/5.52 42.468/5.29 51.287/5.50 78.182/8.50 66.934/4.86

8 27% 13 69% 6 25% 22 46% 12 69% 21 49% 30 51% 8 59% 13 88% 13 30% 13 26% 4 32% 17 42% 21 48% 37 76% 36 61% 16 42% 15 36% 36 57% RPLC, Edman sequencing

P61626 Q9NY56, Q9NPH6 Q06830 P32119 P30044 P30041 P18669 P01833 P35232 P30101 Q9NP55 P14618 Q13228 P02787

16.537/9.38 17.831/7.85

9 47% RPLC, Edman sequencing

22.110/8.27 21.761/5.67 22.026/8.85 24.904/6.02 28.673/6.75 83.314/5.59 29.804/5.57 56.732/5.98 26.713/5.42 57.806/7.95 52.313/6.13 77.050/6.81

matched peptides

17

16 9 8 14 12 14 9 24 6 16 17 36

sequence coverage

identified in other fluidsb

b, c a, c, d d

c a, c a

100 100 83.3 100 54.2 100 83.3 100 100 100 72.9 54.2 100 54.2 100

100 54.2 54.2 58.3 58.3 72.9 62.5

a, b, c 100 62.5 100

38%

63% 39% 44% 54% 49% 24% 33% 55% 28% 40% 40% 50%

frequency (%)

72.9

d a, b, c

100 100 66.6 100 100 100 100 100 100 83.3 54.2 54.2 100 100 100 100 83.3 62.5 62.5

a, b, c

62.5

c, d

a, d a, d a, c, d a, b, c a a

d

a, b, c a a, b, c, d

100 62.5 83.3 91.7 87.5 62.5 50 100 100 100 100 100

research articles

Identification of Human Olfactory Cleft Mucus Proteins Table 1 (Continued)

no.

protein name

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82

Serum Albumin (precursor)* Squamous cell carcinoma antigen 1 Stress-70 protein, mitochondrial (precursor) Stress-induced-phosphoprotein 1 Superoxide dismutase [Cu-Zn] T-complex protein 1, epsilon subunit T-complex protein 1, beta subunit Transitional endoplasmic reticulum ATPase Triosephosphate isomerase Tropomyosin alpha 3 chain Tubulin alpha-ubiquitous chain Tubulin alpha-1 chain Tubulin alpha-6 chain Tubulin alpha-8 chain Tubulin beta-2 chain Tyrosine-protein phosphatase 1E Ubiquinol-cytochrome-c reductase complex core I (precursor) Vitamin D-binding protein (precursor)

83

Swiss-Prot accession number

theorical MW (Da)/pI

P02768 P29508 P38646 P31948 P00441 P48643 P78371 P55072 P60174 P06753 P68363 P68366 Q9BQE3 Q9NY65 P07437 Q12923 P31930

69.367/5.92 44.565/6.35 73.680/5.87 62.639/6.40 15.805/5.70 59.671/5.45 57.488/6.01 89.191/5.14 26.538/6.51 32.819/4.68 50.152/4.94 49.924/4.95 49.695/4.96 50.094/4.94 49.671/4.78 276.606/5.99 52.619/5.94

P02774

52.964/5.40

matched peptides

sequence coverage

50 67% 28 65% 18 33% 20 37% 8 68% 23 38% 7 15% 15 23% 17 76% 13 28% 11 28% 10 26% 10 28% 9 19% 21 60% RPLC, Edman sequencing 8 30% 14

43%

identified in other fluidsb

a, b, c, d a d

c d d

frequency (%)

100 100 66.6 62.5 91.7 66.6 62.5 62.5 91.7 91.7 100 100 100 100 100 72.9

d

62.5

a Eight proteins that are bold typed have been detected only through RPLC and Edman sequencing methods; 75 others were at least identified through 2-DE and MALDI-TOF methods. Seven proteins that are asterisked have been identified with both 2-D/MS and RPLC/Edman sequencing approaches. Frequency corresponds to the relative number of times that proteins were identified on 2-DE. No., numbers are those indicating protein spots in Figure 1. b (a) refers to proteins identified in normal human washed nasal mucus by Casado10 (LC-ESI-Q-TOF), Casado et al.11 (LC-nanoESI-Q-TOF), Ghafouri et al.14 (Western-blot, Edman degradation, and MALDI-TOF/PSD), and Lindahl et al.16,17 (2-D, MALDI-TOF and N-term sequencing); (b) refers to proteins identified in normal human tear by Molloy et al.62 (2-DE and N-terminal sequencing), Li et al.63 (LC-ESI, LC-MALDI-TOF, and Q-Trap/Q-TOF); (c) refers to proteins identified in normal human saliva by Vitorino et al.41 (2-DE and MALDI-TOF-TOF), Hu et al.53 (2-DE, HPLC, MALDI-TOF, and LC-ESI-Q-TOF), Ghafouri et al.54 (2-DE and MALDITOF), Hardt et al.55 (2-DE, MALDI-TOF, HPLC-MALDI-TOF, MALDI-TOF-TOF, and MALDI-QTOF), Guo et al.56 (CIEF/nanoRPLC - LTQ Ion Trap), and Hirtz et al.57 (2-DE and MALDI-TOF); (d) refers to the proteins identified in normal human cerebrospinal fluid by Ogata et al.58 (2-DE and nanoLC-LTQ linear ion trap), Finehout et al.59 (2-DE and MALDI-TOF/TOF-MS), Baraniuk et al.60 (LC-Q-TOF), and Yuan and Desiderio61 (2-D and LC-ESI MALDI-TOF).

Figure 2. Representative reversed-phase liquid chromatogram of nasal mucus sampled at olfactory cleft level. Arrows show the position of the fractions in which protein fragments have been sequenced: F1, basic salivary proline-rich protein 4 allele S (no. 18); F2, basic proline-rich peptide P-E (no. 17); F3, antileukoproteinase 1 (bo. 13); F4, lysozyme (no. 52) and cystatin SN (no. 26); F5, lipid-binding-protein 4 (no. 51); F6, serum albumin (no. 66) and odorant-binding protein (no. 53); F7, epithelial membrane protein-2 (no. 29), serum albumin (no. 66), and tyrosine-protein phosphatase 1E (no. 81).

only are written in bold type in Table 1. They include antileukoproteinase 1 (no. 13), basic proline-rich peptide P-E (no. 17), basic salivary proline-rich protein 4 allele S (no. 18), cystatin SN (no. 26), epithelial membrane protein-2 (no. 29), lipidbinding protein 4 (no. 51), human odorant-binding protein (hOBP) (no. 53) and tyrosine-protein phosphatase 1E (no. 81). Figure 2 represents a typical profile of observed chromatogram. All these proteins were identified by Edman sequencing of fragments (see Supporting Information data table) that incited us to hypothesize protein degradation activity in OCM probably related to cell regulation. Three of these proteins exhibit a pI over 9 (nos. 13, 17, and 18), 5 have a MW lower than 20 kDa or

are fragmented (nos. 13, 17, 26, 29, and 53), one has a MW higher than 200 kDa (no. 81), and one is a membrane protein (no. 29), which explains the reason these proteins were difficult to identify by the 2-DE approach, as previously described.35 The low concentration of lipid binding protein 4 (no. 51) has likely prevented its detection by the 2-DE/MALDI-TOF, since the zone corresponding to this protein spot (around MW 67 kDa and pI 4 to 5) has been thoroughly examinated without revealing any other peptide than trypsin autolytic peptides. As Edman sequencing led to the detection of only its mature N-terminal sequence, the lipid binding protein 4 should not undergo any endoproteolytic degradation. Thus, RPLC/Edman sequencing approach allowed us to detect a specific olfactory cleft protein, hOBP (no. 53). As already reported, this protein seems to be subject to degradation in the OCM because of the presence of not only its N-terminal mature sequence, but also two other fragments (see Supporting Information data table). hOBP fragmentation was verified with a Western blot assay using anti-OBP polyclonal antibodies. Although very high quantity of OCM protein was present (15 µg of total protein), no complete hOBP could be detected with the Western blot assay (data not shown). However, this experiment does not exclude that hOBP could be in weak concentration in healthy human OCM by comparison with numerous mammals including cow, pig, rabbit, mouse, rat, and elephant36 described which have an abundant quantity of OBPs. The weak concentration and/or the fragmentation of this protein in the OCM raises serious questions on any possible role that such proteins could play in human olfaction compared with these other mammals. However, even at very low concentration, the OBP could play an important role in olfaction. The zone of the 2-DE corresponding to low MW (