Characterization of the Human Salivary Basic Proline-Rich Protein Complex by a Proteomic Approach Irene Messana,† Tiziana Cabras,† Rosanna Inzitari,‡ Alessandro Lupi,§ Cecilia Zuppi,‡ Chiara Olmi,‡ Maria Benedetta Fadda,† Massimo Cordaro,⊥ Bruno Giardina,‡,§ and Massimo Castagnola*,‡,§ Department of Sciences Applied to Biosystems, Cagliari University, Cagliari, Italy, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Rome, Italy, Institute for the Chemistry of Molecular Recognition, National Research Council (C.N.R.), Rome, Italy, and Institute of Odontoiatric Clinic, Catholic University of Rome, Rome, Italy Received February 16, 2004
Thirteen samples of human normal whole saliva were analyzed by RP-HPLC-ESI-MS and MALDITOF-MS to investigate the basic proline-rich protein complex. Between known basic-PRPs the P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8c), P-H (or IB-4), IB-6, II-2, IB-1, and IB-8a glucosylated were identified, whereas the II-1, IB-7, PA, and D1-A peptides were not detected. Some detected masses not attributable to known basic-PRPs were putatively ascribed to II-2 and IB-1 nonphosphorylated, II-2 and IB-1 missing the C-terminal arginine residue, and the 1-62 fragment of IB-6, named P-J peptide. A correlation matrix analysis revealed a cluster of correlation among all the basic PRPs (apart from the P-B peptide) which is in agreement with their common parotid origin. Keywords: basic proline-rich proteins • human • saliva • mass spectrometry
Introduction The current availability of powerful analytical tools such as bidimensional electrophoresis coupled to MALDI-TOF-MS and HPLC coupled to electrospray MS is improving researcher ability to elucidate the composition of complex protein mixtures, thus permitting the definition of the “normal” protein pattern of physiological fluids and tissues, that can be used as reference for differential diagnosis of pathological states. With regard to whole human saliva, different groups1,2 were recently able to characterize many salivary proteins by bidimensional electrophoresis coupled to MALDI-TOF-MS. Among them, cystatins (S, SA, SN, and D), statherin, albumin, amylases and calgranulin A and B (S100A8 and A9) were identified, as well as immunoglobulin A, prolactin-inducible protein, zincR2-glycoprotein, interleukin-1 receptor antagonist, lipocalin1, apolipoprotein A-1, β2-glycoprotein, glutathione S-transferase P, and fatty acid-binding protein. However, probably due to the limitations of the electrophoretic separation technique, many small salivary proteins and peptides belonging to the class of proline-rich proteins, histatins and defensins, were not evidenced. Reversed-phase HPLC separations are proved * To whom correspondence should be addressed. Prof. Massimo Castagnola, Istituto di Biochimica e Biochimica Clinica, Facolta` di Medicina, Universita` Cattolica, Largo F. Vito, 00168, Roma, Italy. Tel. and/or Fax: ++39-06-3053598. E-mail:
[email protected]. † Department of Sciences Applied to Biosystems, Cagliari University. ‡ Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome. § Institute for the Chemistry of Molecular Recognition, National Research Council (C.N.R.). ⊥ Institute of Odontoiatric Clinic, Catholic University of Rome.
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to be very useful in order to characterize human salivary peptides and proteins soluble in acidic solution.3 The coupling of RP-HPLC separation to electrospray-ion trap mass spectrometry already allowed us to reach a partial characterization of the human salivary cystatin complex.4 The present study is addressed toward the characterization of the salivary basic proline-rich proteins complex by different approaches based on the combined use of HPLC-ESI-MS and MALDITOF-MS. Salivary PRPs are a family of related polypeptides representing the major fraction of total salivary proteins (more than 50% in weight).5 They are divided into three classes, basic PRPs, glycosylated PRPs, and acidic PRPs. Basic and glycosylated (basic) PRPs are the most complex group, expressed by four different genes named PRB1-PRB4 clustered on chromosome 12p13.2. Numerous homologous and unequal crossing-over are present within the tandem repeats of the third exon, producing frequent length polymorphisms. Moreover, multiple PRPs may be produced from the same gene throughout allelic variations, differential splicing and post-translational modifications.6 Kauffmann and colleagues7-10 have identified eleven basic-PRPs and determined the sequence of 10 of them. Saitoh and coll.11-13 and Isemura and coll.14,15 have identified nine basic-PRPs, called from P-A to P-I, and sequenced seven of them. Even though included in the class of basic PRPs, P-B, and P-C peptides are not a direct product of PRB1-PRB4 genes.6,16 P-B peptide is a product of PROL3 gene (PBI) clustered on chromosome 4q13.317 and P-C peptide derives from the cleavage of acidic PRP-1 (or PRP-2 or Pif-S), which are coded by the PRH1-2 genes (chromosome 12p13.2, near to PRB1-PRB4 genes). 10.1021/pr049953c CCC: $27.50
2004 American Chemical Society
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Analysis of Human Salivary Basic-PRPs
The function of this group of human salivary protein is not well established. As tannin-binding proteins they have probably a protective role against the potential deleterious effects of these substances.18 Recently, an unidentified basic PRP was shown to inhibit HIV-I infectivity.19 If confirmed, this antiviral action can offer adequate explanation for their abundance and molecular heterogeneity in the oral cavity. Therefore, the comprehensive analytical approach described in this study can be useful to clarify function and interplay of basicPRPs in the oral cavity. It is a part of an extensive proteomic study carried out in our laboratories in order to define the “normal” protein profile of the adult human whole saliva. The complete assessment of this feature could be also at the basis for a future widespread use of the human oral fluid for diagnostic purposes.
Experimental Section Reagents. All common chemicals and reagents were of analytical grade and were purchased from Farmitalia-Carlo Erba, (Milan, Italy), Merck (Damstadt, Germany) and Sigma Aldrich (St. Louis, MI). Immobilized trypsin L-1-tosylamido2-phenylethyl chloromethyl ketone-treated was obtained from Pierce Biotechnology (Rockford, IL). Cyano-4-hydroxycinnamic acid, sinapinic acid, and MALDI protein and peptide calibration standards were purchased from Bruker Daltonics (Bremen, Germany). Apparatus. The HPLC-ESI-MS apparatus was a ThermoFinnigan (San Jose, CA) Surveyor HPLC connected by a T splitter to a PDA diode-array detector and to Xcalibur LCQ Deca XP Plus mass spectrometer. The mass spectrometer was equipped with an electrospray ion (ESI) source. The chromatographic column was a Vydac (Hesperia, CA) C8 column, with 5 µm particle diameter (column dimensions 150 × 2.1 mm). Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed by the Autoflex Bruker Daltonics apparatus (Bremen, Germany). Sample Collection and Treatment. The whole human saliva was collected between 2 and 4 pm from 13 normal adult informed volunteers according to a standard protocol.3 Collection time was standardized in order to reduce concentration variability connected to ca.dian rhythms of secretion. Acidic solution (0.2% TFA) was immediately added to salivary samples in 1:1 v/v ratio and the solution centrifuged at 8000 ×g for 5 min. After centrifugation, the supernatant was separated from the precipitate and immediately analyzed by HPLC-mass spectrometry. The following solutions were utilized for the reversed-phase chromatography: (eluent A) 0.056% aqueous TFA and (eluent B) 0.050% TFA in acetonitrile-water 80/20 (v/ v). The gradient applied was linear from 0 to 55% in 40 min, at a flow rate of 0.30 mL/min. The T splitter addressed about a flow-rate of 0.20 mL/min toward the diode array detector and a flow-rate of 0.10 mL/min toward the ESI source. The diode array detector was settled at a wavelength of 214-276 nm. Mass spectra were collected every 3 millisecond in the positive ion mode. MS spray voltage was 4.50 kV and the capillary temperature was 220 °C. Seven selected salivary pools containing basic proline-rich proteins were obtained during the chromatographic analysis by separate collection of the eluent deriving from the diode-array detector. The selected pools were analyzed by MALDI-TOF-MS. They were also submitted to tryptic digestion and analyzed both by RP-HPLC-ESI and MALDITOF-MS under the same conditions applied for the analysis of salivary peptides and proteins.
Tryptic Digestion of the HPLC Pools. Tryptic digestions were made according to the enzyme supplier indications (Pierce Biotechnology, Rockford, USA) on one sample. The lyophilized powders from each of the seven pools collected (0.2 mg) were dissolved into 140 µL of the digestion buffer (NH4HCO3, 0.1 mol/L, pH 8.0) and incubated at 37° C for 5 h with 20 µL of immobilized-trypsin. At the end of the digestion, immobilized-trypsin was separated from the solution by centrifugation at 3000 ×g. The solution was lyophilized and submitted to RP-HPLC-ESI-MS and MALDI-TOF-MS analyses. MALDI-TOF Mass Spectrometry. Lyophilized protein and tryptic digest samples were dissolved in 0.1% aqueous TFA solution, mixed with the appropriate matrix solution and deposited onto the stainless steel target of the MALDI instrument, according to the dried droplet method. Sinapinic acid was used for high-molecular weight peptides, whereas R-cyano4-hydroxycinnamic acid for low-molecular weight peptides. Matrix solutions were prepared as saturated solutions of either R-cyano-4-hydroxycinnamic acid or sinapinic acid in acetonitrile/water (50:50, v/v) containing 0.1% TFA. Calibration was performed using peptide (angiotensin I and II, substance P and bombesin) and protein (insulin, ubiquitin I, cytochrome C, and myoglobin) standards. MALDI-TOF mass spectra were acquired with a pulsed nitrogen laser (337 nm) in positive either linear or reflector mode. In linear mode an ion source of 20 kV, a pulsed ion extraction time of 350 ns, a detector gain voltage of 1300 V and a laser frequency of 5 Hz was used. In positive reflector mode, spectra were obtained with an ion source of 19 kV, a pulsed ion extraction time of 150 ns, a detector gain voltage of 1400 V and a laser frequency of 5 Hz. About 400 scans were averaged for each spectrum to improve signal-to-noise ratio. Data Analysis. Deconvolution of averaged ESI mass spectra was automatically performed either by the software provided with the Deca-XP instrument (Bioworks Browser) or by MagTran 1.0.20 Mass values obtained from the analysis were compared with average theoretical values available from the Swiss-Prot (http://www.expasy.ch) and EMBL (http:// www.embl-heidelberg.de) data banks. Mass values obtained from the analysis of tryptic digests were compared with the average theoretical values corresponding to expected basic PRPs tryptic fragments.
Results Salivary samples were mixed immediately after collection in a 1:1 (v/v) ratio with 0.2% aqueous TFA. The acidic treatment, that partly inhibits salivary proteases thus preserving the sample protein composition, is a necessary prerequisite in order to have a protein solution compatible to the following HPLC-ESI-MS analysis. In fact, the use of TFA provided a satisfactory compromise between high ion-pairing strength for chromatographic separation and satisfactory protein ionization for ESI analysis.3,4 The acidic treatment causes the precipitation of several high molecular weight salivary proteins, such as R-amylases, mucins, mieloperoxidases, and lactoferrin.3 However, peptides and proteins pertaining to histatins, basic and acidic PRPs, statherin, cystatins, and defensins are soluble in acidic solution and they can be directly analyzed by RP-HPLC. The total ion current (TIC) and the 214 and 276 nm UV profiles collected during the RP-HPLC analysis of a “normal” whole saliva sample are reported in Figure 1, with the indication of the rough elution zones of the different peptide salivary classes. Among them, this study was devoted to the identification of Journal of Proteome Research • Vol. 3, No. 4, 2004 793
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Figure 1. Typical RP-HPLC profile of the acidic soluble fraction of whole human saliva. For the chromatographic conditions see the Experimental section. The upper lane represents the total ion current (TIC) profile collected by the ion-trap mass spectrometer. The middle and bottom lanes represent the UV profiles detected at 214 and 276 nm, respectively.
the basic-PRP complex, the most heterogeneous salivary protein class. Table 1 reports the experimental masses detected by RP-HPLC-ESI-MS and attributed to basic-PRPs and the corresponding average theoretical values according to the international data banks (Swiss-Prot code) and the frequency of detection determined on 13 normal samples. The number of other human proteins found in the Swiss-Prot data bank having an average mass within 0.05% of our experimental value is reported in parentheses. The II-2 peptide is not reported in the Swiss-Prot data bank. The theoretical mass of this peptide (7609 amu) was computed according to the sequence reported by Kaufmann et al.,10 whereas the mass of glucosylated IB-8a (11894 amu) corresponds to the experimental value determined by Stubbs et al.16 The mass of IB-6 peptide (11517 amu), and of its P-H fragment (5590 amu) corresponds to the variant having Ser instead of Ala at position 63. Identification of basic-PRPs was usually performed by deconvolution of the averaged ESI mass spectra. This strategy is exemplified in Figure 2, which shows the contemporaneous detection of the basic IB-1 and II-2 peptides. Identification of known peptide in the chromatographic pattern was also obtained by the selected ion monitoring (SIM) strategy, searching the multiple m/z values expected for the given peptide/protein along the entire TIC chromatographic pattern. P-B peptide was found by this strategy at an unexpected elution time, immediately after the chromatographic peak pertaining to statherin, in a RP-HPLC range, where the group of salivary cystatins are also eluted (Figure 1). Being a product of a different gene (see introduction), the sequence of P-B peptide shows some peculiarities with respect to other basic PRPs. It contains three tyrosine, three phenylalanine, one leucine, and two isoleucine residues. These features could explain the low peptide polarity and the anomalous chromatographic elution time. Moreover, these characteristics fit well with the 276 nm absorbance displayed by this peptide (Figure 1). Due to similar polarities, the chromatographic separation of the basic-PRPs was characterized by low selectivity. The 794
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majority of the basic-PRP detected was eluted closely in the 12-20 min elution range. The chromatographic profile pertaining to basic PRPs was divided into seven different pools (Figure 3) according to the following evaluations: (i) the chromatographic pattern; (ii) the presence of masses attributable to basic PRPs; (iii) the absence of an absorbance over 240 nm. Two pools (IV and VI) showed a very complex ESI spectrum, as evident in the middle lane of Figure 3 (pool IV). The gray boxes in Figure 3 underline the chromatographic ranges corresponding to these complex profiles. The detection of known peptides was not easy in these zones, even applying the SIM strategy. As shown in Table 1, quite all the known basic PRPs were identified by the different strategies applied. However, PA, D1-A,21 IB-7 (th. mass 5769 amu) and II-1 (or its glycosylated form; Swiss-Prot no. P81489) peptides were never detected. P-E and IB-6 peptides were detected only twice (in thirteen samples analyzed). In Figure 3, an enlargement of the elution range 9.12-19.60 min, which includes pools I-VI is also reported, as an example of the identification performed on one of the thirteen samples analyzed. As evident from the figure, in the sample, as well as in the other analyzed samples, many sporadic unknown masses were evidenced, which were not considered as potential basic PRPs candidates, due to the absence of further adequate identification. However, some new found peptides were included in Table 1. One of them is a peptide corresponding to the mass of 5944 amu. From the literature, the IB-6 peptide (118 aa long) is cleaved at the Arg62 residue generating the P-H (or IB-4) peptide (fragment 63-118 of IB-6, 56 aa, 5590 amu). Until now, the complementary fragment 1-62 was never detected in human saliva. It has a theoretical mass of 5945 amu, well corresponding to the mass value of 5944 amu detected in nine of the samples analyzed. Following the Saitoh nomenclature, we propose to call this basic PRP (theoretical pI: 11.7) P-J peptide. In fact, even though Saitoh, Isemura and coll.13 did not sequence P-G and P-I peptides, their physicochemical properties do not match with those of the peptide of 5944 amu. Moreover, we found in a close proximity of the elution range of the II-2 (exp. mass 7608 amu) and IB-1 peptides (exp. mass 9593 amu) two masses of 7528 and 9512 amu, which were attributed to the corresponding nonphosphorylated forms. The masses of 7452 and 9437 amu, were attributed to II-2 and IB-1 peptides missing the C-terminal arginine (mass difference from the parent peptides 156 amu), respectively, according to MALDI-TOFMS and tryptic digest MS analyses. To verify the attribution performed by ESI-MS data and to increase information on heterogeneous IV and VI pools, eluate of one sample was collected in correspondence of the selected seven chromatographic pools reported in Figure 3. The lyophilized powder from each pool was submitted to MALDI-TOFMS analysis. This strategy was suggested by the potential higher sensitivity of MALDI-TOF-MS with respect to ESI-IT-MS and by its ability to obtain the contemporaneous detection of multiple peptides/proteins in a complex mixture (in a selected mass window). The MALDI-TOF mass spectra of pool V is shown in Figure 4. The masses pertaining to IB-1 and II-2 basic PRPs, as well as those corresponding to the nonphosphorylated forms and to the forms missing the C-terminal arginine are evident. Table 2 reports other masses (with their frequency) detected in the seven selected pools not attributable to any known PRP and the number of human proteins present in Swiss-Prot data bank having a theoretical averaged mass within
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Analysis of Human Salivary Basic-PRPs
Table 1. Experimental Masses [M] Deteted in the Seven Selected Chromatographic Pools by RP-HPLC-ESI-MS and MALDI-TOF-MS Compared to the Theoretical values [M] of Human Salivary Basic-PRPs Reported in International Data Banks HPLC pool
peptide
Swiss-Prot code
th. massa
exp. ESI
exp. MAL.
freq.
I II II II II III III III V V V-VI V-VI V-VI V-VI VII
P-C (IB-8b) P-F (IB-8c) P-J (IB-6 fr. 1-62) P-E (IB-9) P-D (IB-5) P-H (IB-4) (IB-6 fr. 63-118) IB-8a (glucosylated)b IB-6 IB-1 II-2 IB-1 nonphosph. II-2 nonphosph. IB-1 missing C-term Arg II-2 missing C-term Arg P-B
P02810 P02812 P04280 P02811 P02813 P04280
4371 (0) 5843 (0) 5945 (1) 6024 (0) 6950 (1) 5590 (0) 11894 (1) 11517 (2) 9593 (0) 7609 (1) 9513 (1) 7529 (1) 9437 (0) 7453 (1) 5793 (1)
4371 5843 5944 6024 6950 5590 11898 11519 9593 7608 9512 7528 9437 7452 5793
4369 5842 5944 n.d 6950 5589 n.d. n.d 9592 7607 9510 7524 9435 7452 5793
13/13 9/13 9/13 2/13 11/13 9/13 3/13 2/13 12/13 13/13 6/13 7/13 3/13 7/13 13/13
P04280 P04281 P04281 P04281 P02814
a In parentheses, the number of other human proteins present in Swiss-Prot data bank, with the theoretical average masses within a range of 0.05%, is reported. b Experimentally determined by Stubbs et al.16
Figure 2. ESI mass spectrum (middle panel) obtained by the average of 22 mass spectra collected in 17.91-18.39 min range during the HPLC separation reported in the upper panel (TIC profile). The bottom panel reports the deconvolution of the ESI-MS of the middle panel. Some mass values are slightly different from the mean values reported in Table 1.
a range of 0.05%. These unknown peptides show a polarity similar to basic PRPs and according to their UV absorbance they have low content of aromatic amino acid residues such as PRPs. Even though these properties are not sufficient to include them in the PRPs complex, these mass values should be taken into account for future PRPs searches. To confirm peptide attribution, the lyophilized powder derived from the seven selected pools of Figure 3 was submitted also to tryptic digestion. Although basic PRPs possess few cleavage sites for common proteases (i.e., trypsin and chymotrypsin), an exhaustive simulation provided evidence that the analysis of tryptic digests was enough to exclude the attribution to other human proteins with a mass comprised within a 0.05%. The digests were newly analyzed by HPLC-ESI-MS and
MALDI-TOF-MS. Table 3 reports the average theoretical masses that should be obtained after tryptic digestion of the basic PRPs, compared to the corresponding masses observed in the proper chromatographic pool by ESI and MALDI-TOF mass spectrometry. Quite all the masses corresponding to the tryptic fragments of the basic PRPs recognized in the HPLCESI-MS analysis were detected. However, by MALDI-TOF-MS analysis the 1-91 fragment of IB-1 showed a mass corresponding to the peptide hydrolyzed at N-terminus pyroglutamic moiety (9027 instead of 9010 amu). Small quantities of the fragments 1-93 of IB-1 and 1-72 of II-2 were also detected (masses 9253 and 7269 amu, respectively) suggesting a not complete cleavage for these peptides at arginines 91 and 70, respectively. The experimental mass of 5412 amu is in a Journal of Proteome Research • Vol. 3, No. 4, 2004 795
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Figure 3. TIC profile showing the ranges considered for the collection of the seven pools where salivary basic PRPs were detected (upper panel). The gray boxes correspond to regions showing very complex ESI mass spectrum, such as that of pool IV reported in the middle panel. The bottom panel reports an enlargement of the chromatographic region corresponding to pools I-VI with the principal attributions. In this region, masses attributed to histatins 3, 5, and 6 (His3, His5, and His6) were also detected. Some mass values are slightly different from the mean values reported in Tables 1 and 2. Table 2. Principal Unidentified Experimental Masses [M] Found in the Basic-PRP HPLC Elution Range HPLC pool
exp. mass ESI (freq.)
exp. mass MALDI
na
II III III IV-V VII VII
10434 (6/13) 29415 (2/13) 23461 (11/13) 9060 (2/13) 5215 (2/13) 5062 (11/13)
10434 n.d. n.d. n.d. n.d. 5063
1 1 4 0 0 0
a Number of human proteins present in Swiss-Prot data bank, with the theoretical average masses within a range of 0.05%.
Figure 4. MALDI-TOF-MS analysis (sinapinic acid) of pool V of Figure 3 (for the conditions see Experimental section). The experimental masses reported correspond to the [M+H]+ values.
satisfactory agreement with the theoretical one expected for the fragment of P-J peptide (5415 amu). Surprisingly, the unidentified protein with the mass of 23461 amu reported in Table 2 was not cleaved by trypsin. Figure 5 reports the deconvolution analysis of HPLC peak eluted in the 14.97-15.89 min range of the tryptic digest of pool III showing the intact protein. This property strongly suggests that this unknown protein may be a putative basic PRPs candidate. 796
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With regard to quantitative analysis, the unavailability of proper standards did not allow to determine absolute values of PRP concentration. However, the total ion current of peptides and proteins of interest measured on the deconvoluted spectra was used for their relative quantification. Since the total ion current depends not only from concentration but also from the charge of the analyte, sample treatment was standardized both in dilution and HPLC-ESI-MS procedures, to ensure similar bias, namely same ion suppression, pH and organic solvent effects on the analyte charge. Under these conditions, the total ion current can be roughly considered proportional to protein concentration, and it can be used to evidence correlations existing among different peptides in different samples. On this assumption, a linear least-squares regression analysis was performed among all the couples of ion current values obtained in the samples analyzed. The most frequent basic-PRPs, principal acidic PRPs, statherin and the two potential candidates for basic PRPs (10 434 and 23 461
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Analysis of Human Salivary Basic-PRPs
Table 3. Experimental Masses [M] Detected by RP-HPLC-ESI-MS and MALDI-TOF-MS in the Tryptic Digests of the Selected Seven Chromatographic Pools of Figure 4 Compared to the Theoretical Values [M] peptide
P-B P-C (IB-8b) (a-PRP fr. 106-150) P-D (or IB-5) P-E (or IB-9) P-F (or IB-8c) IB-6 P-H (or IB-4) P-J IB-1 IB-1 nonphosph. II-2 II-2 nonphosph. IB-8ab (glucosylated)
a
theor.
theor. tryptic masses
ex. mass ESI
ex. mass MALDI
5793 4371
3103-2131-328-285 4371
3103-2132 4370
3105-2132 4372
6950 6024 5843 11517 5590 5945 9593 9513 7609 7529 11712 (11894)
6950 5440-358-261 5441-261-176 5590-5415-548 5590 5415-548 9009-358-261 (9252)a 8930-358-261 7027-358-261 (7270)a 6947-358-261 5526-35541895-548-261
6950 5441 5441 5590 5590 5412 9010 (9253)a 8930 7025 (7269)a 6948 5527 3554 1896
6951 5441 5441 5590 5590 5413 9027 (9270)a n.d. 7026 (7268)a n.d. 5527 3555 1895
Mass values of not-cleaved fragments 1-92 of IB-1 and 1-72 of II-2. b Mass computed on the basis of the gene sequence16 (see discussion).
Figure 5. HPLC-ESI-MS profile of the tryptic digest of pool III. Upper panel: TIC profile. Middle panel: average (38 spectra) ESI mass spectrum corresponding to the peak labeled in the upper panel. Bottom panel: deconvolution of the ESI mass spectrum reported in the middle panel.
amu, respectively) were considered in this statistical analysis. Proteins observed in a reduced number of samples, such as P-E, IB-6, and IB8-a glucosylated, were not included in the analysis. Figure 6 reports as an example the correlation analysis performed for the II-2/10 434 and IB-1/P-J couples. The array of correlation coefficients is reported in Table 4. The statistic significance (p) was determined on the basis of the correlation coefficient and the number of the couples utilized for the correlation.
Discussion The different mass spectrometric approaches utilized in this study permitted to identify in the RP-HPLC pattern of “normal” human whole saliva the following known salivary basic PRPs. They are P-B, P-C (or IB-8b), P-D (or IB-5), P-E (or IB-9), P-F (or IB-8c), P-H (or IB-4), IB-6, II-2, IB-1, and IB-8a glucosylated (Table 1). All these attributions cannot be ascribed to other human proteins with a mass compatible (( 0.05%) present in the Swiss-Prot data bank for the following reasons: (i) the Journal of Proteome Research • Vol. 3, No. 4, 2004 797
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Figure 6. Correlation analysis performed on the ionic current of II-2 peptide vs that one of protein with a mass of 10 434 amu and on the ionic current of IB-1 vs that one of P-J peptide.
physicochemical properties of these proteins do not match with the polarity and UV absorbance detected; (ii) the experimental pattern of the tryptic peptides do not correspond to the expected one for these proteins. For the same reasons, the masses of Table 2 cannot be ascribed to none human protein with a mass compatible (( 0.05%) present in the Swiss-Prot data bank, even though the mass of proteins from data bank do not take into account either processing or post-translational modifications. The sensitivity of the analysis permitted also to putatively identify the nonphosphorylated forms of II-2 and IB-1 peptide, until now not detected in human saliva. The two mass values of 7452 and 9437 amu, detected in pool VI containing II-2 and IB-1 peptides were putatively recognized as the corresponding peptides missing the C-terminal arginine. The results of the tryptic digestion of pool VI were in a good agreement with this interpretation. In fact, since trypsin digestions provide the cleavage of small C-terminal peptides, identical fragments for the parent proteins were observed. The putative identification of the fragment 1-62 deriving from the IB-6 peptide, until now not identified, was carried out. In this respect, it should be outlined that during the determination of the structure of P-H peptides, Saitoh and coll.13 isolated also two peptides named P-G and P-I, whose sequence was not determined. However, the reported SDSPAGE results indicated that P-G peptide should have a mass lower and P-I a mass sensibly greater than P-H peptide. Therefore, the fragment 1-62 should not correspond neither to P-G nor to P-I peptides. According to Saitoh nomenclature the new peptide was called P-J. The other known basic PRPs were not detected along the complete chromatographic pattern. They include IB-7 peptide (th. mass 5769 amu), II-1 peptide and the two fragments of P-B peptide, namely the P-A peptide (th. mass 3798 amu) and the D1-A peptide (th. mass 1435 amu). This lack can be due either to a peptide concentration lower than the detection limit of the method or to several post-translational modifications occurred on the peptides. However, we cannot exclude that these basic PRPs could be absent in the samples analyzed or that they could be eluted in the complex chromatographic range corresponding to pools IV and VI of Figure 3, where the detection of any peptide is troublesome. The same explanation can be invoked for the lacking of detection of any glycosylated basic PRPs. The apo-P-O peptide (Swiss-Prot code P10163) has a theoretical mass of 23 498, very 798
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close to the unidentified 23 461 mass (found in 11 of 13 samples) observed in pool III (Table 2). However, whereas the digestion of apo-P-O peptide should provide principal tryptic peptides with a theoretical mass of 13 498, 6968, and 2511 amu, surprisingly the protein of 23 461 mass remained intact after tryptic digestion (Figure 5). The masses expected from the digestion of P-O were not found in the tryptic digest of pool III. The few studies performed on glycosylated basic PRPs22-26 described a very heterogeneous glycosylation pattern. The presence of highly heterogeneous glycosylated-proteins may be the principal reason of the complex ESI spectrum observed in pools IV and VI (see Figure 3). This hypothesis will be the aim of further studies. The analysis of the tryptic fragments carried out by HPLCESI-MS and MALDI-TOF-MS and reported in Table 3 confirmed satisfactorily the identity of all the basic PRPs detected in this study. We found in the tryptic digest of pools II and III a fragment of 5527 amu, which could derive both from IB-8a and IB-7. Since the mass corresponding to IB-7 was not found, this fragment probably derives only from IB-8a glucosylated. Stubbs and coll.16 determined by mass spectrometry for this last protein a mass of 11 894(( 3) amu. They also established the presence of a mole of glucose/ mole protein. As a consequence, the mass of the apo-protein should correspond to 11 732(( 3) amu. We found on pool III by MALDI-TOF-MS a mass of 11 731 amu (not reported in Table 1) which is very close to this value. However, this mass value is not in agreement with that one expected on the basis of the gene sequence16 (11 712 amu; ∆mass ) 20 amu), implying an error in the gene sequence determination. The simulation of the tryptic digestion was performed on the basis of the gene sequence (Table 3). Fragments of 5527, 3554, and 1896 amu were found corresponding to those expected on the basis of gene sequence. Therefore, sequence conflict and glucosylation should be confined to the small 58-62 and 120-121 fragments, which are not easily detectable by the techniques utilized in this study. The complete sequence of II-1 is not known10 due to single amino acid uncertainness at the 172 residue. On the basis of the known sequence, it is expected that this basic PRP should be quite insensitive to tryptic digestion, and it should generate theoretically the fragment 1-169 from a protein of 174 amino acids residues. The mass of 17222 amu corresponding to this fragment was not found in any pool of the tryptic digest. Several not attributable masses were detected in the HPLC pools collected. Those observed only in a single sample were considered without statistical significance. The masses observed in more than one sample are reported in Table 2, together with their frequency. The best candidates to be putatively included in the basic PRP class are the proteins with mass values of 10 434 and 23 461 amu, due to their chromatographic behavior, frequency, lacking of aromatic absorbance and significant correlation with the other basic PRPs (Table 4). Moreover, the protein with a mass of 23 462 amu, likewise other basic PRPs, is not sensible to tryptic digestion. In this table, only peptides/ proteins showing high frequency were included (see Tables 1 and 2). P-B, P-C, and II-2 peptides were found in all the thirteen samples analyzed. Concerning other basic-PRPs not found in all the samples, our results suggest that none may be considered vicarious for the others in human saliva. Rather, some samples are contemporaneously lacking of several PRPs. In this respect, it is noteworthy to observe two distinctive clusters of significant correlations in Table 4. The first cluster is repre-
research articles
Analysis of Human Salivary Basic-PRPs
Table 4. Correlation Coefficients Obtained from Least Square Linear Correlations among the Ionic Current Measured for the Most Frequent Basic PRPs, Acidic PRP-1, PRP3, and Statherin Identified by HPLC-ESI-MS PRP1
PRP3
P-B
P-C
P-D
P-J
P-F
P-H
IB-1
II-2
23462
10434
b
PRP3
0.772
-
P-B
0.436
0.828
a
a
a
P-C
0.626
0.576
0.634
-
P-D
0.083
0.160
0.456
0.752
c
b a
P-J
0.232
0.128
0.340
0.629
c
0.904 b
P-F
0.367
P-H
0.317
0.372
0.069
0.199 0.178
0.118
0.757
a
a
0.640
0.644
a
IB-1
0.099
0.158
0.327
0.551 b
II-2
0.074
0.115
0.327
0.730 a
23462
0.221
0.053
0.342
0.616
c
0.921 c
0.826 c
0.903
c
0.913
-
c
0.845 c
0.936
0.121 b
0.799
c
0.912 c
0.968
c
0.821 c
0.427 c
0.848
0.928 b
0.796
c
0.857 c
0.945 c
10434
0.067
0.163
0.546
c
0.916 c
a
0.269
0.526
0.512
0.473
0.029
0.816
0.949
0.664
-
0.415
0.415
0.183
0.297
0.084
0.147
0.224
0.261
0.430
a
Stath a
0.147
0.489
0.649
p < 0.05; p < 0.01; p < 0.001. The masses of 23 462 and 10 434 amu pertain to two proteins basic PRPs candidates. b
c
sented by the group of classical basic PRPs (including the unidentified proteins with a mass of 10 434 and 23 461 amu) that showed multiple highly significant correlations, suggesting a common prevalent parotid secretion.7,10,27 The second is represented by the acidic PRP and PB peptide, according to their prevalent submandibular secretion.17,21 The P-C peptide show a correlation of low statistical significance with both the acidic and basic PRPs groups, whereas statherin did not show a highly significant correlation with any salivary peptide pertaining either to acidic or basic PRPs classes, according to a peculiar secretion pathway.19,27-29 A role for basic PRPs in the oral cavity, except for their ability to bind deleterious tannins,18 has not been defined. Recently, it was shown that an unidentified basic PRP inhibited HIV-I infectivity.19 If confirmed, this antiviral activity could open new insight on one possible role of this complex group of peptides/ proteins specific to saliva, having such a very peculiar sequence and structure.30 We hope that future possible correlations existing between different pathologies and anomalous presence or absence of basic-PRPs in the oral cavity might offer some elucidation on their roles. This study is a preliminary step toward this trend. As final conclusion, it should be remarked that, even though the principal classes of salivary components are today quite completely established, the results of this study, as well as the results of recent studies performed by other groups,1,2 indicate that protein and peptide composition of “normal” adult human saliva is extremely complex and far to be completely defined. Moreover, an experimental approach suitable for the contemporaneous detection of the complete pattern until now does not exist. For instance, the proteomic approach described in this study did not allow for the obtaining of any information on the peptides and proteins that precipitate in acidic solution (mucins, amylases), which are of great relevance in the oral cavity. Moreover, as shown in Figure 3, several complex ranges of the chromatographic pattern of human salivary proteins cannot be adequately characterized by the
ESI-MS approach adopted. On the other hand, proteomics approaches based on bidimensional electrophoresis followed by MALDI-TOF-MS analysis1,2 seem to be unable to evidence small salivary peptides, which cannot be properly detected in the electrophoretic separation. A great lot of work will be necessary in order to develop an adequate analytical approach and to adequately identify the “entire” human salivary protein pattern. Abbreviations: proline-rich proteins: PRPs.
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