Top-down HPLCâESIâMS detection of - American Chemical Society

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Top-down HPLC−ESI−MS detection of S‑Glutathionylated and S‑Cysteinylated Derivatives of Cystatin B and Its 1−53 and 54−98 Fragments in Whole Saliva of Human Preterm Newborns Federica Iavarone,† Tiziana Cabras,‡ Elisabetta Pisano,§ Maria Teresa Sanna,‡ Sonia Nemolato,§ Giovanni Vento,∥ Chiara Tirone,∥ Costantino Romagnoli,∥ Massimo Cordaro,⊥ Vassilios Fanos,§ Gavino Faa,§ Irene Messana,‡ and Massimo Castagnola*,† †

Istituto di Biochimica e di Biochimica Clinica, Università Cattolica and/or Istituto per la Chimica del Riconoscimento Molecolare, CNR, Istituto Scientifico Internazionale (ISI) Paolo VI, Roma, Italy ‡ Dipartimento di Scienze della Vita e dell’Ambiente and §Dipartimento di Scienze Chirurgiche, Università di Cagliari, Cagliari, Italy ∥ Istituto di Clinica Pediatrica and ⊥Istituto di Clinica Odontostomatologica, Università Cattolica, Roma, Italy ABSTRACT: Analysis by a HPLC−ESI−MS top-down proteomic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated, S-cysteinylated, and S−S 2-mer (on Cys3) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of Smodified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1−53 and 54−98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1−53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development. KEYWORDS: saliva, preterm, proteomics, top-down, cystatin B, S-glutathionyl, S-cysteinyl and gastric cancer.6,7 A lower cystatin B expression level was observed in tumor tissue compared to that of the corresponding normal tissue in esophageal carcinoma and prostatic adenocarcinoma.8,9 Cystatin B presence was also revealed in both neurons and glial cells differentiated from neural stem cells and in hippocampal cultures with a different localization inside the different cell types.10 Cystatin B inhibits bone resorption in rats by decreasing intracellular cathepsin K activity.11 In humans cystatin B allows overactivity of cathepsin K in patients with Unverricht-Lundborg disease.12 Cathepsin K overactivity in turn leads to altered bone remodelling through the higher bone turnover component.12 Recently, by an integrated top-down/bottom-up proteomic approach,13,14 our group evidenced that cystatin B is present in human adult whole saliva mostly as S-glutathionylated, S-cysteinylated (on the Cys3 residue), and S−S 2-mer derivatives.15 Moreover, as described in the present study, noticeable amounts

1. INTRODUCTION Cystatins are natural tight-binding reversible competitive inhibitors of cysteine proteinases. They are involved in various biological processes and are widespread in all living organisms where they regulate proteolysis and take part in the molecular mechanisms underlying various pathologies.1 Human cystatin A and B belong to the family 1 of the cystatin superfamily. They are nonglycosylated proteins of ∼11 kDa lacking the signal sequence and disulfide bonds and are generally expressed intracellularly.2 Type 1 cystatin inhibitory activity is vital for the delicate regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteinases such as the papain (C1) family, including cysteine cathepsins L, S, and H.3 Failures in biological mechanisms controlling proteinase action result in many diseases such as neurodegeneration, osteoporosis, arthritis, and cancer.4 Cystatin B regulates the degradation of FLIPL and thereby the TNF-related apoptosis-inducing ligand in melanoma cells.5 Furthermore, the activity of cystatin B has been reported in several human carcinomas, such as human colorectal carcinoma © 2013 American Chemical Society

Received: October 12, 2012 Published: January 2, 2013 917

dx.doi.org/10.1021/pr300960f | J. Proteome Res. 2013, 12, 917−926

Journal of Proteome Research

Article

Figure 1. Series of three graphs utilized for determination of cystatin A (panels A), S-unmodified cystatin B (panels B), cystatin B (panels C), Sglutathionylated cystatin B (panels D), and S-cysteinylated cystatin B (panels E) in one typical sample of preterm newborn whole saliva. The left panels show the extracted ion current (XIC) peak utilized for the detection of each component (m/z values of Table 1). The peak corresponding to the S−S 2mer cystatin B is that eluting at 34.08 min in panel C(XIC). The peak at 33.1 min of panel C(XIC) corresponds to S-unmodified cystatin B, because the odd m/z values utilized for the XIC search of the 2-mer are the same used for the XIC search of S-unmodified 1-mer. The central panels show the ESI spectra collected on the peaks of the left panels. The panel DE(ESI) shows simultaneously the ESI spectra of S-glutathionylated and S-cysteinylated cystatin B, because the two derivatives elute together (panels D(XIC) and E(XIC)). The right panels show the average mass of each component obtained by deconvolution of the ESI spectra.

2. MATERIALS AND METHODS

of the fragments 1−53 and 54−98 of cystatin B, as well as the S-derivatives of fragment 1−53, were revealed in human preterm newborn saliva. The purpose of the present study was the determination and the characterization by a top-down approach of the relative amounts of the different forms of cystatin B and its fragments in human saliva of preterm newborns as a function of postconceptional age (PCA) and the comparison of the relative amounts in subjects with different ages. The results of this study highlight specific enzymatic activities in the oral cavity of preterm newborns, which can be a clue to specialized pathways working during fetal oral development.

2.1. Ethics Statements

The study protocol and written consent forms were approved by both the Pediatric Department Ethics Committee and the Medical Ethics Committee of the Faculty of Medicine of the Catholic University of Rome (according to the instructions of the Declaration of Helsinki). Informed consent forms were filled out by parents of babies, and all rules have been complied with. 2.2. Reagents and Apparatus

Chemicals and reagents, all of LC−MS grade, were purchased from J. T. Baker (Deventer, The Netherlands), Merck 918



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Table 1. Mav, Elution Time, and Multicharged Ions Selected as Extracted Ion Current Peaks (XIC) of Cystatin A, Cystatin B (N-Terminally Acetylated) and Its S-Glutathionyl, S-Cysteinyl, and S−S 2-mer Derivatives; Fragment 1−53 and Its S-Derivatives, and Fragment 54−98 in Preterm Newborn Saliva protein or fragment (Swiss-Prot code)

elution time (min)

exptl Mav (theor Mav)

cystatin B (P04080)

32.6−33.4

cystatin B S-glutathionyl

32.4−33.1

cystatin B S-cysteinyl

32.5−33.2

cystatin B S−S 2-mer

33.6−34.4

cystatin B Fr. 1−53

29.3−30.1

cystatin B Fr. 54−98

29.2−30.0

cystatin B Fr. 1−53 S-glutathionyl

28.3−29.1

cystatin B Fr. 1−53 S-cysteinyl

28.4−29.2

cystatin B Fr. 1−53 S−S 2-mer cystatin B + Fr. 1−53 S−S hetero 2-mer cystatin A (P01040)

undetectable undetectable 31.3−32.1

11182 ± 2 (11181.6) 11487 ± 2 (11486.7) 11301 ± 2 (11300.7) 22361 ± 3 (22361.2) 5843 ± 1 (5842.6) 5357 ± 1 (5357.0) 6148 ± 1 (6147.7) 5962 ± 1 (5961.7) (11683.2) (17022.2) 11006 ± 2 (11006.5)

multicharged ions selected as XIC peaks; m/z (charge) 1017.5 (11+), 1119.2 (10+), 1243.4 (9+), 1398.7 (8+), 1598.4 (7+), 1864.6 (6+) 1045.3 (11+), 1149.7 (10+), 1277.3 (9+), 1436.9 (8+), 1642.0 (7+), 1915.5 (6+) 1028.3 (11+), 1131.1 (10+), 1256.6 (9+), 1413.6 (8+), 1615.4 (7+), 1884.5 (6+) 973.2 (23+), 1017.4 (22+), 1965.8 (21+), 1119.0 (20+), 1177.9 (19+), 1243.3 (18+), 1316,4 (17+), 1398.6 (16+), 1491.8 (15+), 1598.2 (14+), 1721.1 (13+), 1864.4 (12+) 1169.5 (5+), 1461.5 (4+), 1948.6 (3+) 1072.4 (5+), 1340.3 (4+), 1786.7 (3+) 1025.7 (6+), 1230.6 (5+), 1537.7 (4+) 1193.4 (5+), 1491.2 (4+), 1988.3 (3+) 974.6 (12+), 1063.1 (11+), 1169.3 (10+), 1299.1 (9+), 1461.4 (8+), 1670.0 (7+) 1135.8 (15+), 1216.9 (14+), 1310.4 (13+), 1419.5 (12+), 1548.5 (11+), 1703.2 (10+) 1001.6 (11+), 1101.7 (10+), 1224.0 (9+), 1376.8 (8+), 1573.4 (7+), 1835.4 (6+)

Figure 2. Series of three graphs utilized for the determination of S-unmodified fragment 1−53 of cystatin B (panels A), fragment 1−53 of Sglutathionylated cystatin B (panels B), fragment 1−53 of S-cysteinylated cystatin B (panels C), and fragment 54−98 of cystatin B (panels D) in one sample of preterm newborn whole saliva. The left panels report the extracted ion current (XIC) peak utilized for the detection of each component (m/z values of Table 1). The central panels show the ESI spectra collected on the peaks of the left panels. The panel BC(ESI) shows simultaneously the ESI spectra of fragments 1−53 of S-glutathionylated and S-cysteinylated cystatin B, because the two derivatives elute together (panels B(XIC) and C(XIC)). The right panels show the average mass of each derivatives obtained by deconvolution of the ESI spectra. 919



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Table 2. Experimental and Theoretical [M + H]+ Monoisotopic Values Obtained by Analysis of the Deconvoluted MS/MS Spectra Carried out by LTQ Orbitrap XL on the Four-Charged Ion with an m/z Value of 1461.48, Corresponding to the N-Terminally Acetylated Fragment 1−53 of Cystatin Ba exptl [M + H] 5666.83 5535.80 5432.78 5375.76 5304.73 5207.68 5049.61 4948.57 4820.50 4723.44 4652.41 4551.37 4480.33 3685.97 2845.53 1942.04 1470.76 1300.66 1066.55 a

+

theor [M + H] 5666.83 (y52) 5535.80 (y51) 5432.79 (y50) 5375.76 (y49) 5304.73 (y48) 5207.68 (y47) 5049.61 (y45) 4948.56 (y44) 4820.50 (y43) 4723.45 (y42) 4652.41 (y41) 4551.36 (y40) 4480.33 (y39) 3685.97 (y32) 2845.52 (y25) 1942.04 (y18) 1470.76 (y14) 1300.65 (y12) 1066.55 (y10)

+

theor [M + H] 465.13 (b4) 536.17 (b5) 4902.44 (b44) 5216.60 (b47) 5315.66 (b48) 5386.70 (b49) 5658.81 (b52)

+

exptl [M + H]

Table 3. Experimental and Theoretical [M + H]+ Monoisotopic Values Obtained by Analysis of the Deconvoluted MS/MS Spectra Carried out by LTQ Orbitrap XL on the Hepta-Charged ion with an m/z Value of 766.26, Pertaining to the Fragment 54−98 of Cystatin Ba

+

465.13 536.17 4902.43 5216.60 5315.63 5386.70 5658.81

Only the fragments of the b and y series detected are reported.

(Darmstadt, Germany), and Sigma-Aldrich (St. Louis, MO, USA). The HPLC−ESI-IT-MS apparatus was a Surveyor HPLC system (ThermoFisher, San Jose, CA, USA) connected by a T splitter to a PDA diode-array detector and to an LCQ Deca XP Plus mass spectrometer. The mass spectrometer was equipped with an ESI source. The chromatographic column was a Vydac (Hesperia, CA, USA) C8 column, with 5 μm particle diameter (column dimensions 150 mm × 2.1 mm). Some samples of whole saliva were also analyzed by an Ultimate 3000 Micro HPLC apparatus (Dionex, Sunnyvale, CA, USA) equipped with a FLM-3000-Flow manager module coupled to an LTQ Orbitrap XL apparatus (ThermoFisher). In this case a Zorbax SB300 C8 (Agilent) column (3.5 μm particle diameter; column dimension 150 mm × 1.0 mm) was utilized.

a

exptl [M + H]+

theor [M + H]+

theor [M + H]+

exptl [M + H]+

5207.66 5094.58 4966.50 4867.43 4730.35 4631.29 4574.27 4459.25 4330.19 4215.18 4068.10 3969.03 2988.48 2891.43 2754.39 2625.33 2383.19 2173.06 2072.01 1958.92 1871.90 1594.77 1123.54 924.41 329.15

5207.67 (y44) 5094.59 (y43) 4966.49 (y42) 4867.42 (y41) 4730.36 (y40) 4631.29 (y39) 4574.27 (y38) 4459.25 (y37) 4330.20 (y36) 4215.18 (y35) 4068.11 (y34) 3969.04 (y33) 2988.49 (y25) 2891.43 (y24) 2754.37 (y23) 2625.33 (y22) 2383.19 (y20) 2173.06 (y18) 2072.01 (y17) 1958.92 (y16) 1871.89 (y15) 1594.79 (y13) 1123.54 (y9) 924.41 (y7) 329.15 (y2)

488.32 (b4) 625.38 (b5) 724.45 (b6) 896.50 (b8) 1025.54 (b9) 1140.57 (b10) 1287.64 (b11) 1386.70 (b12) 2167.14 (b18) 2254.18 (b19) 2367.26 (b20) 2601.37 (b22) 2730.41 (b23) 2844.46 (b24) 2972.55 (b25) 3069.61 (b26) 3182.69 (b27) 3283.74 (b28) 3396.82 (b29) 3483.85 (b30) 3597.90 (b31) 3760.96 (b32) 3889.02 (b33) 3990.07 (b34) 4104.11 (b35) 4303.24 (b37) 4431.34 (b38) 4683.42 (b40) 4812.46 (b41) 4925.55 (b42) 5026.59 (b43) 5189.66 (b44)

488.32 625.38 724.45 896.50 1025.54 1140.57 1287.64 1386.71 2167.14 2254.17 2367.26 2601.37 2730.41 2844.46 2972.55 3069.61 3182.69 3283.74 3396.82 3483.85 3597.90 3760.95 3889.04 3990.07 4104.13 4303.23 4431.34 4683.43 4812.46 4925.55 5026.60 5189.66


(1) 6 full-term infants (3 females and 3 males) with a saliva collection within a week from birth. The babies were born after uncomplicated pregnancies with normal birth and no clinical problems and admitted to the Policlinico “A. Gemelli” nursery. Their mean ± SD gestational age and birth weight were 272 ± 7 days (38 ± 1 weeks) and 3280 ± 150 g, respectively. (2) 21 pediatric subjects (8 females, 13 males) with an age range from 1 to 5 years. (3) 20 healthy adults (10 males, 10 females) with an age range from 25 to 58 years. After collection salivary samples were immediately mixed with an equal volume of 0.2% TFA (v/v) in an ice bath. After stirring, the acidic solution was centrifuged at 9000g for three min to remove the precipitate, and the acidic clear solution was either immediately analyzed by HPLC−ESI−MS (100 μL, corresponding to 50 μL of saliva) or stored at −80 °C until analysis.

2.3. Subjects Enrolled and Sample Collection and Treatment

Fifteen newborns (7 males, 8 females) with a birth weight ranging between 500 and 1250 g and PCA between 27 and 31 weeks (193−217 days) admitted to the Neonatal Intensive Care Unit (NICU) were enrolled for this study. Infants with major congenital malformations or prenatal infections were excluded. Sample collection was performed on the same preterm newborn during several weeks after birth at established time intervals (1 or 2 weeks). When possible, it was also performed after discharge from the neonatal unit, during the periodical check visits within about 1 year follow-up. Saliva was not collected when sample collection seemed to cause even a minimal stress to the newborn and/or to his/her parents. For these reasons, sample collection was more frequent during the hospital stay and sporadic thereafter. In this way we were able to analyze 87 saliva specimens from newborns ranging from 191 to 545 days of PCA. Whole saliva samples from the following groups of subjects were also studied:

2.4. RP-HPLC−ESI−MS Analysis

The following solutions were utilized for the chromatographic separation: (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 a flow rate of about 0.20 mL/ 920



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Table 4. Experimental and Theoretical [M + H]+ Monoisotopic Values Obtained by Analysis of the Deconvoluted MS/MS Spectra Carried out by LTQ Orbitrap XL on the Four-Charged Ion with an m/z Value of 1537.75 Pertaining to the N-Terminally Acetylated and S-Glutathionylated (Cys3) Fragment 1−53 of Cystatin Ba

a

Table 5. Experimental and Theoretical [M + H]+ Monoisotopic Values Obtained by Analysis of the Deconvoluted MS/MS Spectra Carried out by LTQ Orbitrap XL on the Four-Charged Ion with an m/z Value of 1491.23 Pertaining to the N-Terminally Acetylated and S-Cysteinylated (Cys3) Fragment 1−53 of Cystatin Ba

exptl [M + H]+

theor [M + H]+

theor [M + H]+

exptl [M + H]+

exptl [M + H]+

theor [M + H]+

theor [M + H]+

exptl [M + H]+

5971.91 5432.79 5375.78 5304.73 5207.66 5049.61 4948.57 4820.50 4652.40 4551.36 4351.29 4122.18 3985.12 3872.03 3800.99 3685.97 3302.45 2716.48 2588.39 2345.30 2217.21 2089.12 1942.05 1598.86 1470.78 1399.72 1300.65 1213.62 1066.55 938.46 525.23

5971.91 (y52) 5432.79 (y50) 5375.77 (y49) 5304.73 (y48) 5207.68 (y47) 5049.61 (y45) 4948.56 (y44) 4820.50 (y43) 4652.41 (y41) 4551.36 (y40) 4351.28 (y38) 4122.18 (y36) 3985.12 (y35) 3872.03 (y34) 3801.00 (y33) 3685.97 (y32) 3302.74 (y29) 2716.48 (y24) 2588.39 (y23) 2345.30 (y21) 2217.21 (y20) 2089.12 (y19) 1942.04 (y18) 1598.85 (y15) 1470.76 (y14) 1399.72 (y13) 1300.65 (y12) 1213.62 (y11) 1066.55 (y10) 938.46 (y9) 525.23 (y5)

770.20 (b4) 841.23 (b5) 1096.36 (b8) 1197.40 (b9) 1325.46 (b10) 3171.40 (b27) 3429.51 (b29) 3557.58 (b30) 3686.62 (b31) 3928.76 (b33) 4056.85 (b34) 4675.20 (b39) 4845.31 (b41) 5079.41 (b43) 5422.60 (b46) 5521.66 (b47) 5620.73 (b48) 5691.77 (b49) 5849.84 (b51) 5963.88 (b51)

770.20 841.23 1096.35 1197.40 1325.46 3171.39 3429.48 3557.58 3686.64 3928.77 4056.86 4675.21 4845.32 5079.44 5422.59 5521.66 5620.72 5691.77 5849.85 5963.88

5785.83 5654.80 5432.78 5375.76 5304.72 5207.67 5049.60 4948.56 4820.50 4652.40 4551.36 4122.17 3872.02 3685.97 2845.52 2217.21 1942.05 1470.79 1399.72 1300.65 1213.62 1066.55 624.30

5785.84 (y52) 5654.80 (y51) 5432.79 (y50) 5375.77 (y49) 5304.73 (y48) 5207.68 (y47) 5049.61 (y45) 4948.56 (y44) 4820.50 (y43) 4652.41 (y41) 4551.36 (y40) 4122.18 (y36) 3872.03 (y34) 3685.97 (y32) 2845.52 (y25) 2217.21 (y20) 1942.04 (y18) 1470.76 (y14) 1399.72 (y13) 1300.65 (y12) 1213.62 (y11) 1066.55 (y10) 624.30 (y6)

655.17 (b5) 1139.40 (b10) 3114.37 (b28) 3243.42 (b29) 3742.69 (b33) 3870.79 (b34) 4659.25 (b41) 4893.35 (b43) 5021.44 (b44) 5236.53 (b46) 5335.60 (b47) 5505.71 (b49) 5663.78 (b51)

655.17 1139.40 3114.37 3243.43 3742.72 3870.86 4659.24 4893.36 5021.42 5236.51 5335.62 5505.69 5663.77

a


2.5. Data Analysis

Deconvolution of averaged ESI mass spectra was automatically performed by the Xcalibur 2.0.7 software or by MagTran 1.0 software.16 Experimental mass values were compared with average theoretical values available at the Swiss-Prot data bank (http://us.expasy.org/tools), where cystatin B and cystatin A have the accession numbers P04080 and P01040, respectively. The identification of cystatin B, its derivatives, and fragments was accomplished as described in the Results section. The relative abundance of cystatin A, cystatin B, and its derivatives was determined by considering the eXtracted Ion Current (XIC) peak area and relating it to 1.0 mL of saliva. The value [XIC peak area]/mL of saliva is linearly proportional to the peptide concentration and can be used to monitor relative abundances, under constant analytical conditions.17 In determining the XIC peak area the right choice of m/z values for the detection of the protein of interest is needed. This is to avoid choosing m/z ESI potentially overlapping spectra belonging to other proteins that elute very close in crowded areas of chromatographic elution, as described in Results section. The window for all of these values was in a range of ±0.5 m/z. The percentage error of the measurements was less than 10%.


min toward the diode array detector and 0.10 mL/min toward the ESI source. During the first 5 min of separation the eluate was not addressed to the mass spectrometer to avoid instrument damage due to the high salt concentration. The diode array detector was set at a wavelength of 214 and 276 nm. Mass spectra were collected every 3 ms in the positive ion mode. MS spray voltage was 4.50 kV, and the capillary temperature was 250 °C. High-resolution HPLC−ESI−MS/MS experiments were performed using a LTQ Orbitrap XL apparatus, using the following eluents for reverse-phase chromatography: 0.056% aqueous TFA (eluent A) and 0.050% TFA in acetonitrile−water 80/20 (v/v) (eluent B). The applied gradient was 0−4 min 5% B, 4−38 min from 5% to 50% B (linear), 38−41 min from 50% to 90% B (linear), at a flow rate of 80 μL/min. Mass spectra were collected in data dependent scan (MS/MS data) mode with a capillary temperature of 250 °C, a sheath gas flow of 18 arbitrary unities, a source voltage of 3.6 kV, and a capillary voltage of 40 V. Measurements were performed in the positive ion mode, and mass accuracy was calibrated before measurements. Selected protein charge states were isolated with a width of m/z 6−10 and activated for 30 ms using 35% normalized collision energy and an activation q of 0.25.

3. RESULTS 3.1. Detection in Preterm Newborn Saliva of Cystatin B, Cystatin B Fragments, and S-Glutathionyl and S-Cysteinyl Derivatives

The presence of cystatin B (N-terminally acetylated) and its S-glutathionyl, S-cysteinyl, and S−S 2-mer derivatives in preterm 921



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Figure 3. (Left) Values of the [XIC area]/mL of saliva (arbitrary unit) of S-unmodified, S-glutathionylated, S-cysteinylated, and S−S 2-mer cystatin B measured in 87 samples of preterm newborn whole saliva, plotted as a function of days of PCA. (Right) Percentages of S-unmodified, S-glutathionylated, S-cysteinylated, and S−S 2-mer cystatin B measured in 87 samples of preterm newborn whole saliva, plotted as a function of days of PCA.

Since the only cysteine residue of cystatin B is located in the third position, we also searched in the chromatographic TIC profile the masses of the glutathionylated (Mav 6147.7 Da) and the cysteinylated (Mav 5961.7 Da) derivatives of fragment 1−53 (Table 1). Two peptides with Mav 6148 ± 1 Da and 5962 ± 1 Da were detected in the elution range 28.7 ± 0.5 min. The HPLC− ESI−MS analysis of several preterm newborn saliva samples treated with DTT highlighted the disappearance of the two peptides and the concomitant increase of the peak corresponding to the unmodified fragment 1−53 (5843 Da) in agreement with the presence of a S-modification. Manual inspection of high resolution HPLC−ESI−MS/MS spectra confirmed the structural attributions as S-glutathionyl and S-cysteinyl derivatives of N-terminally acetylated fragment 1−53 (Tables 4 and 5). The MS/MS fragments y51 (Table 5) and y52 (Table 4 and 5), as well as all the MS/MS fragments of the b series, were diagnostic for the unambiguous characterization of the S-modification. We have also searched by XIC procedures and deconvolution of ESI spectra the masses corresponding to the S−S 2-mer of the 1−53 fragment of cystatin B (theor Mav 11683.2 Da; Table 1), as well as the potential S−S 2-mer deriving from intact cystatin B

newborn saliva was established on the basis of their experimental average mass (Mav) and elution times, as previously reported for adult saliva (Figure 1, Table 1).15 To confirm the protein identity, several samples of preterm and at-term newborn saliva were treated with DTT and analyzed by HPLC−ESI−MS immediately after reduction. After the treatment, the masses attributed to the S-glutathionyl (Mav 11487 ± 2 Da), the S-cysteinyl (Mav 11301 ± 2 Da), and the S−S 2-mer (22361 ± 3 Da) derivatives of cystatin B were not detected in any sample, while the intensity of the cystatin B (11182 ± 2 Da) peak increased proportionally. Moreover, in the chromatographic profiles of saliva from preterm newborns two peptides with Mav of 5843 ± 1 Da and 5357 ± 1 Da eluting at 29.7 ± 0.4 and 29.6 ± 0.4 min, respectively, were constantly detected (Figure 2). Manual inspection of the high-resolution MS/MS fragmentation spectra carried out on the ion at 1461.48 m/z (charge +4) generated by the 5843 Da peptide and on the ion at 766.26 m/z (charge +7) generated by the 5357 Da peptide allowed their unambiguous identification as the fragments 1−53 (N-terminally acetylated; 5843 Da) and 54−98 (5357 Da) of cystatin B, respectively. Attributions of MS/MS data are reported on Tables 2 and 3. 922



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of PCA), a group of 21 babies from 1 to 5 years old, and a group of 20 adults. The [XIC peak area]/ mL of saliva of cystatin A was also measured to verify potential correlations with the levels of cystatin B. Figure 3 (left) shows that the level of cystatin B and its S-derivatives decreased significantly as a function of PCA. However, the shapes of the decrease were not similar. The level of unmodified cystatin B decreased faster than that of the S-derivatives, as evident in Figure 3 (right), which shows the percentages of the different cystatin B derivatives as a function of PCA. Taken together the data highlight that in the first period of extra-uterine life the level of all forms of cystatin B decreased as a function of PCA, but the percentage of S-derivatives increased. The comparison of the mean values of [XIC peak area]/mL of saliva of cystatin B and its S-derivatives measured in three preterm newborns groups with different PCA, in at-term newborns, in babies 1 to 5 years old, and in adults (Figure 4) showed that the salivary levels of S-derivatives of cystatin B in preterm newborns after normal term of delivery were not significantly different from those in at-term newborns, babies, and adults. In the adult, S-unmodified cystatin B was usually not detectable in whole saliva.15 3.3. Determination of XIC Peak Area/mL of Saliva of Cystatin B Fragments and S-Derivatives of Fr. 1−53 as a Function of PCA

The values of the [XIC peak area]/mL of saliva of the fragment 1−53 (S-unmodified plus S-glutathionyl and S-cysteinyl derivatives) and the fragment 54−98 measured on preterm newborn whole saliva as a function of PCA are reported in Figure 5. The fragments were detected in 12 out of 15 preterm newborns. The three subjects without fragments were three twin sisters born at 203 days of PCA (6 months and 20 days). Fr. 1−53 was detectable in 66 samples, and Fr. 54−98 in 71 samples (out of 87), corresponding always to the first samples of each series. It is evident from the figure that the fragments levels are highly dependent on the PCA, disappearing at about 275 days of PCA (corresponding to the days of PCA of full term newborns). The fragments were not detectable in whole saliva of at-term newborns, nor in whole saliva of pediatric subjects and adults. Figure 6 (left) shows that the levels of the S-unmodified fragment 1−53 of cystatin B do not match with its derivatives. As observed for intact cystatin B, the S-unmodified Fr. 1−53 represents the predominant form immediately after birth, and the percentages of the S-glutathionylated and S-cysteinylated derivatives increase as a function of PCA. Because after 270 days of PCA the fragments were usually not detectable, it was impossible to carry out comparisons with the other groups of subjects. Figure 7 shows the mean values of the [XIC peak area]/ mL of saliva (left) and the mean percentages (right) measured in

Figure 4. (Left) Mean values of measured [XIC area]/mL of saliva (arbitrary unit) of S-unmodified, S-glutathionylated, S-cysteinylated, and S−S 2-mer cystatin B measured in the following groups: (i) preterm newborns 176−226 days old (PCA; n = 37); (ii) preterm newborns 227−290 days old (PCA; n = 36); (iii) preterm newborns older than 291 days (PCA; n = 14); (iv) at-term newborns (n = 6); (v) babies 1 to 5 years old (n = 21); (vi) adults (n = 20). (Right) mean percentages of S-unmodified, S-glutathionylated, S-cysteinylated, and S−S 2-mer cystatin B measured in the same groups. (Student’s t test: * p < 0.05; ** p < 0.01)

and its 1−53 fragment (Mav 17022.2 Da; Table 1), without any success. 3.2. Relative Quantification of Cystatin B Derivatives As a Function of PCA

The relative amounts of cystatin B and its S-derivatives, as well as those of its Fr. 1−53 and 54−98, and of the S-derivatives of Fr. 1−53, were determined in 87 samples collected from 15 preterm newborns at different days of PCA, and the data were compared with those obtained in a group of 5 at-term newborns (275 days

Figure 5. Values of [XIC area]/mL of saliva (arbitrary unit) of the sum of the S-unmodified, S-glutathionylated, and S-cysteinylated derivatives of fragment 1−53 of cystatin B (left; n = 66) and of fragment 54−98 of cystatin B (right; n = 71) plotted as a function of days of PCA. 923



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Figure 6. (Left) Values of [XIC area]/mL of saliva (arbitrary unit) of S-unmodified, S-glutathionylated, and S-cysteinylated fragment 1−53 of cystatin B measured in 66 samples of preterm newborn whole saliva, plotted as a function of days of PCA. (Right) Percentages of S-unmodified, S-glutathionylated, and S-cysteinylated fragment 1−53 of cystatin B measured in 66 samples of preterm newborn whole saliva, plotted as a function of days of PCA.

three different groups: (i) preterm 176−226 days old (PCA; n = 29); (ii) preterm 227−290 days old (PCA; n = 33); (iii) preterm older than 291 days old (PCA; n = 4). The S−S dimer of Fr. 1−53 and the S−S heterodimer deriving from a disulfide bridge binding the entire cystatin B and Fr. 1−53 were searched in all of the samples but were never detected.

suggested that the cleavage event is successive to the Smodification of Cys3 residue. A significant, although not very high, correlation was observed between the levels of cystatin B versus both S-unmodified Fr. 1− 53 and the total amount of Fr. 1−53 (all forms). The correlation between the levels of cystatin B versus Fr. 54−98 and between the levels of S-glutathionylated and S-cysteinylated cystatin B and the corresponding S-derivatives of Fr. 1−53, although lower, was significant. Taken together, the correlations suggested that the two events are independent, i.e., the cleavage is not influenced by the S-modification of the protein. Table 6 also reports the very significant correlation found between the level of total and S-unmodified cystatin B versus cystatin A, suggesting that the two proteins play a cooperative role in the development of the preterm newborn oral cavity.

3.4. Correlations among the Different Derivatives of Cystatin B and between Cystatin A and B

The coefficient of the correlation analyses (Pearson r) among the values of the [XIC peak area]/mL of saliva for the different cystatin B components and its fragments are reported in Table 6. Very significant correlations (p < 0.01) were found for the following analyses: (a) levels of S-glutathionylated versus S-cysteinylated cystatin B, (b) levels of Fr. 54−98 versus Fr. 1− 53 (total), and (c) percentages of S-unmodified, S-glutathionylated, and S-cysteinylated cystatin B versus the corresponding derivatives of Fr. 1−53. In analysis (a) the positive relationship suggests that the two events of S-modification of the Cys3 residue are related. This suggestion is reinforced by the significant correlation found between the S-glutathionylated and S-cysteinylated derivatives of Fr. 1−53. In analysis (b) the expected positivity shows that the fragments are generated by a specific cleavage and that they are not prone to extensive further cleavages. In fact, in the naturally occurring salivary proteome of preterm infants relevant amounts of other fragments deriving from cystatin B were not found, both by manual and program-assisted (Proteome Discoverer) searches. In analysis (c) the correlations were highly positive both including and excluding the values of the [XIC peak area]/ mL of saliva of the S−S 2-mer of cystatin B. This positivity

4. DISCUSSION Results of this study confirm, as previously reported,14,15 that during the development of the oral cavity preterm newborns require levels of cystatin A and B about 2 orders of magnitude greater than adults and that these two proteinase inhibitors decrease their levels at a PCA corresponding to that of at-term newborns. In consideration of the highly significant correlation of their levels, cystatins A and B probably play cooperative roles during fetal development. Moreover, the detection of Sderivatives of cystatin B in preterm newborns suggests other functions of this protein in the oral environment. The low percentage of S-derivatives of cystatin B measured in preterm newborns (especially at low PCA) with respect to pediatric subjects and adults may suggest a low activity of the enzyme(s) responsible for the S-modification in fetuses. 924



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protein is mainly under the control of glutaredoxin (Grx), which can act as a glutathionylating enzyme under an oxidative stimulus and as a deglutathionylase when the oxidative stress subsides.18 No significant differences were observed in the levels of S-derivatives by comparing saliva of preterm newborns treated and not treated with oxygen and artificial ventilation. This finding suggests that during fetal development oxidative stress conditions do not affect the inducible enzyme(s) involved in the S-modification. While little information is available on the significance of the S-cysteinylation,19 S-glutathionylation is a well-known protein modification. For many proteins, glutathionylation affects function, and the reversible glutathionylation of specific proteins has been implicated in regulation of cellular homeostasis in health and disease.20,21 The presence of two fragments of cystatin B (1−53 and 54−98) generated by the cleavage Y↓F highlights the activity of a very specific oral proteinase in preterm newborns. The results of this study suggested that S-modification of cystatin B occurs before the cleavage and that the two events are independent, i.e., the cleavage is not influenced by the Smodification of the protein. The absence of fragments of S−S 2-mer cystatin B suggested that only the monomeric protein is substrate of the proteinase. A cleavage between tyrosine and phenylalanine residues (Y↓F) complies with a chimotrypsin-like enzymatic activity, but the strict specificity suggests a very specific consensus sequence for the cleavage. A search of the Merops (http://merops.sanger.ac.uk/) data bank returned various possibilities other than chimotrypsin A, such as chimosin, cathepsin E, metalloproteinase 2, ADAMTS4, endothelin converting enzyme 1, and some peptidases of the chimase class (mast cell chimotrypsin-like proteinase). Whatever enzyme is involved, it is not inhibited by cystatin B and A, because the levels of the proteins correlated significantly with those of the fragments. Obviously, from these data it is impossible to establish if cystatin B is a natural substrate of the enzyme, thereby implying a functional role for the fragments, or rather if the fragments observed are byproducts, without any functional meaning, of a proteinase, whose activity is devoted to other specific fetal oral cleavage processes.

Figure 7. (Left) Mean values of [XIC area]/mL of saliva (arbitrary unit) of S-unmodified, S-glutathionylated, and S-cysteinylated derivatives of fragments 1−53 of cystatin B measured in the following groups: (i) preterm newborns 176−226 days old (PCA; n = 29); (ii) preterm newborns 227−290 days old (PCA; n = 33); and (iii) preterm newborns older than 291 days (PCA; n = 4). (Right) Mean percentages of S-unmodified, S-glutathionylated, and S-cysteinylated derivatives of fragments 1−53 of cystatin B measured in the same groups. (Student’s t test: * p < 0.05; ** p < 0.01)

Nonetheless, the oral cavity environment of the fetus not experienced with high oxygen levels could be at the basis of the difference observed. Glutathionylation is an oxidative posttranslational modification that occurs on some cysteine residues under basal conditions for some proteins, while for others it is a transient modification arising during oxidative stress.18 Reversible S-glutathionylation of

Table 6. Correlation Coefficient (Pearson r) Computed between Cystatin A, the Different Derivatives of Cystatin B, Fragment 54−98, and the S-Derivatives of Fragment 1−53 of Cystatin B unmod cyst B S-glut cyst B S-cyst cyst B S−S 2-mer cyst B unmod Fr. 1−53 cyst B S-glut Fr. 1−53 cyst B S-cyst Fr. 1−53 cyst B total Fr. 1−53 cyst B

0.391 0.381 0.334 0.533

S-glut cyst B 0.735 0.398

S-cyst cyst B

unmod Fr. 1−53 cyst B

S-glut Fr. 1−53 cyst B

Fr.54−98 cyst B

0.584

0.256 0.082

0.652 0.557

0.713

0.478

0.923 0.401

Fr. 54−98 cyst B

0.354

cyst A

0.721

0.267 0.858 cystatin B total ↑ % unmod cyst B % S-glut cyst B % S-cyst cyst B

% unmod Fr. 1−53 cyst B ↓ 0.694

% S-glut Fr. 1−53 cyst B ↓

% S-cyst Fr. 1−53 cyst B ↓

0.715 0.530 925



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(14) Castagnola, M.; Inzitari, R.; Fanali, C.; Iavarone, F.; Vitali, A.; Desiderio, C.; Vento, G.; Tirone, C.; Romagnoli, C.; Cabras, T.; Manconi, B.; Sanna, M. T.; Boi, R.; Pisano, E.; Olianas, A.; Pellegrini, M.; Nemolato, S.; Heizmann, C. W.; Faa, G.; Messana, I. The surprising composition of the salivary proteome of preterm human newborn. Mol. Cell. Proteomics 2011, 10 (1), M110.003467. (15) Cabras, T.; Manconi, B.; Iavarone, F.; Fanali, C.; Nemolato, S.; Fiorita, A.; Scarano, E.; Passali, G. C.; Manni, A.; Cordaro, M.; Paludetti, G.; Faa, G.; Messana, I.; Castagnola, M. RP-HPLC−ESI−MS evidenced that salivary cystatin B is detectable in adult human whole saliva mostly as S-modified derivatives: S-glutathionyl, S-cysteinyl and S-S 2-mer. J. Proteomics 2012, 75 (3), 908−913. (16) Zhang, Z.; Marshall, A. G. A universal algorithm for fast and automated charge state deconvolution of electrospray mass-to-charge ratio spectra. J. Am. Soc. Mass Spectrom. 1998, 9 (3), 225−233. (17) Levin, Y.; Schwarz, E.; Wang, L.; Leweke, F. M.; Bahn, S. Labelfree LC-MS/MS quantitative proteomics for large-scale biomarker discovery in complex samples. J. Sep. Sci. 2007, 30 (14), 2198−2203. (18) Mieyal, J. J.; Gallogly, M. M.; Qanungo, S.; Sabens, E. A.; Shelton, M. D. Molecular mechanisms and clinical implications of reversible protein S-glutathionylation. Antioxid. Redox Signaling 2008, 10 (11), 1941−1988. (19) Dalle-Donne, I.; Rossi, R.; Colombo, G.; Giustarini, D.; Milzani, A. Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends Biochem. Sci. 2009, 34 (2), 85−96. (20) Chu, F.; Ward, N. E.; O’Brian, C. A. PKC isozyme S-cysteinylation by cystine stimulates the pro-apoptotic isozyme PKC delta and inactivates the oncogenic isozyme PKC epsilon. Carcinogenesis 2003, 24 (2), 317−325. (21) Lim, S. Y.; Raftery, M. J.; Goyette, J.; Geczy, C. L. SGlutathionylation regulates inflammatory activities of S100A9. J. Biol. Chem. 2010, 285 (15), 14377−14388.

Conclusively, this study confirms that in the oral cavity of preterm infants the activity of some enzymes has not still reached that one measured in the adult. On the contrary different and specific enzymes, probably involved in the development, which either decrease or disappear in the adult age, are active in the fetal oral cavity and its annexes. Future studies will be necessary to establish their effective functional meaning.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: ++39-06-3053598. E-mail: massimo.castagnola@ icrm.cnr.it. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of the Nando Peretti Foundation, Università di Cagliari, Università Cattolica in Rome, MIUR, Italian National Research Council (CNR), Regione Sardegna.

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REFERENCES

(1) Zavasnik-Bergant, T. Cystatin protease inhibitors and immune functions. Front. Biosci. 2008, 13, 4625−4637. (2) Ochieng, J.; Chaudhuri, G. Cystatin superfamily. J. Health Care Poor Underserved 2010, 21 (1 Suppl), 51−70. (3) Abrahamson, M.; Barrett, A. J.; Salvesen, G.; Grubb, A. Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentration in body fluids. J. Biol. Chem. 1986, 261 (24), 14313−14320. (4) Turk, V.; Stoka, V.; Turk, D. Cystatins: biochemical and structural properties and medical relevance. Front. Biosci. 2008, 13, 5406−5420. (5) Yang, F.; Tay, K. H.; Dong, L.; Thorne, R. F.; Jiang, C. C.; Yang, E.; Tseng, H. Y.; Liu, H.; Christopherson, R.; Hersey, P.; Zhang, X. D. Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIP(L) from degradation by the E3 ligase itch in human melanoma cells. Cell Death Differ. 2010, 17 (8), 1354−1367. (6) Sheahan, K.; Shuja, S.; Murnane, M. J. Cysteine protease activities and tumor development in human colorectal carcinoma. Cancer Res. 1989, 49 (14), 3809−3814. (7) Plebani, M.; Herszènyi, L.; Cardin, R.; Roveroni, G.; Carraro, P.; Paoli, M. D.; Rugge, M.; Grigioni, W. F.; Nitti, D.; Naccarato, R.; Farinati, F. Cysteine and serine proteases in gastric cancer. Cancer 1995, 76 (3), 367−375. (8) Shiraishi, T.; Mori, M.; Tanaka, S.; Sugimachi, K.; Akiyoshi, T. Identification of cystatin B in human esophageal carcinoma, using differential displays in which the gene expression is related to lymphnode metastasis. Int. J. Cancer 1998, 79 (2), 175−178. (9) Mirtti, T.; Alanen, K.; Kallajoki, M.; Rinne, A.; Soderstrom, K. O. Expression of cystatins, high molecular weight cytokeratin, and proliferation markers in prostatic adenocarcinoma and hyperplasia. Prostate 2003, 54 (4), 290−298. (10) Brännvall, K.; Hjelm, H.; Korhonen, L.; Lahtinen, U.; Lehesjoki, E.; Lindholm, D. Cystatin-B is expressed by neural stem cells and by differentiated neurons and astrocytes. Biochem. Biophys. Res. Commun. 2003, 308 (2), 369−374. (11) Laitala-Leinonen, T.; Rinne, R.; Saukko, P.; Väan̈ änen, H. K.; Rinne, A. Cystatin B as an intracellular modulator of bone resorption. Matrix Biol. 2006, 25 (3), 149−157. (12) Korja, M.; Kaasinen, V.; Lamusuo, S.; Marttila, R. J.; Parkkola, R. Hyperostosis frontalis interna as a novel finding in Unverricht-Lundborg disease. Neurology 2007, 68 (13), 1077−1078. (13) Messana, I.; Inzitari, R.; Fanali, C.; Cabras, T.; Castagnola, M. Facts and artifacts in proteomics of body fluids. What proteomics of saliva is telling us? J. Sep. Sci. 2008, 31 (11), 1948−1963. 926


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