Proteomic Study of Pilocytic Astrocytoma Pediatric Brain Tumor

Article pubs.acs.org/jpr

Proteomic Study of Pilocytic Astrocytoma Pediatric Brain Tumor Intracystic Fluid Ilaria Inserra,†,# Federica Iavarone,†,# Claudia Martelli,† Luca D’Angelo,†,‡ Daniela Delfino,† Diana Valeria Rossetti,† Gianpiero Tamburrini,‡ Luca Massimi,‡ Massimo Caldarelli,‡ Concezio Di Rocco,‡ Irene Messana,§ Massimo Castagnola,†,∥ and Claudia Desiderio*,∥ †

Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Università Cattolica del Sacro Cuore, Rome 00168, Italy Unità Complessa di Neurochirurgia Infantile, Istituto di Neurochirurgia - Policlinico Universitario A. Gemelli, Largo Agostino Gemelli 8, Rome 00168, Italy § Dipartimento di Scienze della Vita e dell’Ambiente, Università di Cagliari, Monserrato (Cagliari), Italy ∥ Istituto di Chimica del Riconoscimento Molecolare − UOS Roma, Consiglio Nazionale delle Ricerche, Largo F. Vito 1, Rome 00168, Italy ‡

ABSTRACT: Liquid chromatography in coupling with highresolution ESI-LTQ-Orbitrap mass spectrometry was applied for a proteomic study of pediatric pilocytic astrocytoma brain tumor intracystic fluid by an integrated top-down/bottom-up platform. Both of the proteomic strategies resulted complementary and support each other in contributing to a wide characterization of the protein and peptide content of the tumor fluid. Top-down approach allowed to identify several proteins and peptides involved in different biological activities together with the characterization of interesting proteoforms such as fibrinopeptide A and its truncated form, fibrinopeptide B, complement C3f fragments, β-thymosin peptides, ubiquitin, several apolipoproteins belonging to A and C families, apolipoprotein J and D, and cystatin C. Of particular interest resulted the identification of a N-terminal truncated cystatin C proteoform, likely involved in immune response mechanism modulations and the identification of oxidized and glycosylated apolipoproteins including disulfide bridge dimeric forms. The bottom-up approach confirmed some of the experimental data findings together with adding the characterization of high-molecular-mass proteins in the samples. These data could contribute to elucidate the molecular mechanisms involved in onset and progression of the disease and cyst development. KEYWORDS: proteomics, brain tumors, pylocitic astrocytoma, intracystic fluid, mass spectrometry

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INTRODUCTION Pilocytic astrocytoma is the most common pediatric brain tumor, accounting for 20% pediatric population brain tumors with an incidence ranging between 0.5 and 0.6/100 000 person/year.1 Pilocytic astrocytoma is classified as a World Health Organization (WHO) grade I tumor, and it occurs in patients aged from a few months up to 20 years, with a peak of incidence between 6 and 9 years, without sex predilection. Among pediatric astrocytomas, pilocytic astrocytoma is of lower grade of malignancy and therefore it is generally associated with a more favorable prognosis. Pilocytic astrocytoma can be solid, cystic, or pseudocystic, and the cystic wall is formed by the neoplastic cells. The cysts can be monolocular or multilocular within the tumor nodule. The intracystic fluid is yellowish or brownish due to its high protein concentration or, rarely, due to a previous hemorrhage or necrosis. The instrumental diagnosis, obtained by head computed tomography (CT) and brain magnetic resonance (MR) with contrast, allows studying the morphological characteristics of the lesion and its relationship © 2014 American Chemical Society

with the surrounding structures: all of this information is fundamental for surgical planning but not for the definitive diagnosis of the nature of the tumor, finally obtained by postoperative histopathological examination. Gross local resection, when possible, is the treatment of choice. Local recurrence after gross total resection occurs in 10 to 20% of patients, and the 10 year overall survival rate is as high as 96%. The pathogenesis of pilocytic astrocytoma is not understood yet. Therefore, the proteomic characterization of the tumor intracystic fluid could provide an interesting tool for the comprehension of molecular mechanisms involved in tumorigenesis and for the identification of potential therapeutic targets. Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: August 1, 2014 Published: September 25, 2014 4594

dx.doi.org/10.1021/pr500806k | J. Proteome Res. 2014, 13, 4594−4606

Journal of Proteome Research

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MS characterization and successfully allows the identification of the parent high-molecular-weight protein on the basis of the MS detection of its proteotypic peptides.

A small number of papers in literature have been devoted to proteomic studies in relation to pediatric brain tumor diseases, mainly reporting data obtained from tryptic digests of adult patient samples, and very few are related to pilocytic astrocytoma. A gene expression study identified Apolipoprotein D as a potential diagnostic marker for discriminating the low-grade malignant pilocytic astrocytoma from the rapidly proliferating anaplastic astrocytoma tumor.2 Mass-spectrometry-based proteomics studies on pilocytic astrocytoma were performed on cerebrospinal fluid (CSF)3,4 and brain tumor tissues.5−7 2-D gel electrophoresis in coupling with MALDI-TOF/TOF MS of adult CSF samples evidenced a differential protein expression associated with different astrocytoma tumor grades,3,4 identifying gelsolin3 and specific higher tumor grades4 potential biomarkers. The first and extensive proteomic study on pediatric pilocytic astrocytoma appeared in 2011 using 2-DE in coupling with MALDI-TOF.5 In this study, the integration of proteomics, genomics, and bioinformatics platforms allowed the characterization of numerous protein species characteristic of low-grade pilocytic astrocytoma tissue in comparison with normal tissue, outlining the importance of evaluating the differential expression of entire groups of proteins rather than single or small species. Another very recent paper on brain tumor tissue identified the differential expression of several proteins and miRNA in pediatric astrocytomas of different grades by MALDI-TOF MS of 2-DE digested spots and microarray analysis mainly focusing on vimentin, calreticulin and 14−3−3 epsilon hub protein interactome.6 The analysis of microdissected cells from brain biopsies of astrocytoma by nano-LC in coupling with linear ion trap MS after protein digestion evidenced a significantly different protein expression between the diverse tumor grades, particularly relevant in the most aggressive ones.7 Previous papers from our group applied topdown LC−MS proteomics to study CSF and tumor intracystic fluid in relation to different pediatric brain tumors.8−11 The proteomic analysis of CSF samples from patients affected by posterior cranial fossa tumors, including pilocytic astrocytoma, evidenced the potential role of prognosis biomarkers for specific hemoglobin fragments, the LVV- and VV-hemorphin7.8 The study of intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor by top-down/bottom-up proteomic platform resulted in the identification of protein and peptides involved in inflammation, mineralization processes, cell motility, and lipid transport,9−11 some of them in common with the pilocytic astrocytoma intracystic fluid present findings. To the best of our knowledge, no proteomics studies have been to date reported on pilocytic astrocytoma intracystic fluid, the object of the present work. This study therefore presents the first LC−MS proteomic investigation of pilocytic astrocytoma pediatric brain tumor intracystic fluid by the application of top-down/bottom-up integrated approaches. The two proteomic strategies are complementary and differently contribute to proteome and peptide characterization with their exclusive features. The topdown approach enables the characterization of proteins in their entire state, making possible the identification of isoforms and post-translation modifications (PTMs). This approach is particularly suitable for low-molecular-mass proteins and peptides. Conversely, the bottom-up approach, the most widely used, analyzes proteins after enzymatic digestion prior to LC−

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EXPERIMENTAL SECTION

Reagents

Iodoacetamide (IA), D,L-dithiothreitol (DTT), ammonium bicarbonate powder, and trypsin (for proteomics analysis) were purchased from Sigma-Aldrich (St. Louis, MO). Trifluoroacetic acid (TFA) and formic acid (FA) were obtained from Fluka (Sigma-Aldrich Chemie, Buchs, Switzerland) and J.T. Baker (Deventer, Holland), respectively. Chloroform (RPE grade) and acetonitrile (ACN) were purchased, respectively, from Prolabo (Fontenay-sous-Bois, France) and Sigma-Aldrich. All organic solvents used were of LC−MS grade. Ultrapure water was obtained from P.Nix Power System apparatus, Human, Seoul, Korea. Apparatus

HPLC-ESI-MS2 analysis was performed on a high-resolution LTQ Orbitrap XL mass spectrometry apparatus (ThermoFisher) with ESI source in coupling with an Ultimate 3000 Micro HPLC (Dionex, Sunnyvale, CA) equipped with a FLM3000-Flow manager module. Protein and peptide separation was performed on different Zorbax (Agilent Technologies, Santa Clara, CA) chromatographic columns, 300SB-C8 (3.5 μm, 300 Å pore size, 150 × 1.0 mm) or 300SB-C18 (5 μm, 300 Å pore size, 150 × 1.0 mm), depending on if top-down or bottom-up approach was used, respectively. Procedures

Pilocytic Astrocytoma Cystic Fluid Samples Collection and Treatment. Pilocytic astrocytoma intracystic fluids were collected from four patients (all males, 3−11 years) admitted to Pediatric Neurosurgery Unit of Catholic University of Rome. The samples were collected under sterile conditions during the surgery: after the surgical approach consisting of craniotomy and dural opening, the cystic fluid was obtained before the tumor removal through a needle aspiration under ultrasound (US) view. An aliquot of the collected intracystic fluid was used for the present study with the written consent of their parents. All samples were then immediately stored at −80 °C. Pilocytic astrocytoma cystic fluids underwent a simple pretreatment procedure previously described10,11 that produces the most abundant protein depletion and lipid fraction removal. Briefly, the samples, thawed at room temperature, were acidified by adding 10% volume of 1.0% (v/v) TFA aqueous solution (0.1% TFA final content) and successively added with 2× volumes of ACN, vortex-mixed (1.5 min), and centrifuged (23 791g × 15 min, 4 °C) after 10 min of storage in ice. An aliquot of the resulting supernatant was therefore extracted with 2× volume of chloroform, vortex-mixed (1 min), and centrifuged (12 100g × 2 min, room temperature). The resulting aqueous extracts were directly analyzed by LC−MS for top-down proteomic analysis or successively treated for disulfide bonds reduction or trypsin digestion for bottom-up analysis. The protein concentration was determined in triplicate using the Bradford assay (Bio-Rad Laboratories, Hercules, CA). For disulfide bonds reduction, the extracts were added 1:1 (v/v) with 200 mM DDT solution in 100 mM ammonium bicarbonate buffer pH 8.0 and successively incubated under stirring (5 min at 100 °C, followed by 15 min at 50 °C) and 4595



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Figure 1. Total ion current (TIC) profile (A) of the extracted Pilocytic Astrocytoma pediatric brain tumor intracystic fluid analyzed by HPLC-ESILTQ-Orbitrap-MS top-down approach (for detailed operating conditions see the Experimental Section). (B) Chromatogram enlarged view at retention time range 14−35 min.

from 5 to 55% B (40 min), (ii) from 55 to 100% B (8 min), (iii) from 100% B to 5% (9 min) for a total time of 61 min at a flow rate of 80 μL/min. The injection volume was 20 μL. High-resolution full-scan MS and MS2 spectra were collected in data-dependent scan mode (DDS) with a resolution of 60 000 and 30 000, respectively, in the range from 300 to 2000 m/z. In DDS acquisition mode, the three most intense multiply charged ions were selected in a time window of 3 ms and fragmented by collision-induced dissociation (CID) (35% normalized collision energy). The capillary temperature was 250 °C, source voltage 4 kV, capillary voltage 37 V, tube lens voltage 245 V. Bottom-Up HPLC−MS Analysis. Bottom-up HPLC-ESILTQ-Orbitrap analyses were performed using an aqueous solution of FA (0.1%) as eluent A and ACN/water (80:20, v/v) with 0.1% FA as eluent B. Chromatographic separation was conducted by a three-step gradient: (i) from 5 to 55% of eluent B (42 min), (ii) from 55 to 100% of eluent B (8 min), and (iii) from 100% of eluent B to 5% (2 min) for a total run time of 61 min at a flow rate of 80 μL/min. The injection volume was 20 μL.

then cooled at room temperature. Cysteine residues alkylation was achieved by adding 1:1 (v/v) IA 200 mM in 100 mM ammonium bicarbonate buffer pH 8.0 and 1 h of incubation in the dark at room temperature. The excess of IA was thereafter removed by the addition of 1:1 volume of DDT 200 mM solution in 100 mM ammonium bicarbonate buffer at pH 8.0. In the bottom-up strategy, this procedure was followed by enzymatic digestion with trypsin adding 1:50 (m/m) trypsin/ total protein content. For bottom-up analysis, the samples were not TFA-acidified at the beginning to avoid inhibition of the trypsin enzyme activity. The samples were left to digest overnight at 37 °C, successively acidified with aqueous TFA 0.2% (v/v) to stop the digestion, and immediately frozen and lyophilized. HPLC−MS Analysis

Top-Down HPLC−MS Analysis. Top-down HPLC−MS analyses were performed by means of high-resolution MS apparatus and using aqueous FA (0.1%) as eluent A and ACN/ water (80:20, v/v) with 0.1% FA as eluent B with chromatographic conditions as specified below: applied step gradient: (i) 4596



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Table 1. List of Proteins and Peptides Identified in Pilocytic Astrocytoma Intracystic Fluid Proteomic Analysis by Top-Down Approach Swiss Prot Code

Rt (min)a

Mr (theor.)b

Mr (exp.)b

P02671 P02671 P02675 P01024 P01024 P01024 P63313 P62328 P02654 P02654 B4DV12 P02655 P02656 P02656 P02656 P61769 P01034

fibrinopeptide A truncated fibrinopeptide A fibrinopeptide B complement C3f fragment truncated complement C3f fragment truncated complement C3f fragment thymosin β-10 thymosin β-4 truncated apolipoprotein C I apolipoprotein C I ubiquitin apolipoprotein C II apolipoprotein C III apolipoprotein C III apolipoprotein C III β-2 microglobulin truncated cystatin C

19.92 20.03 21.86 19.39 19.39 19.39 20.30 19.80 39.70 39.78 30.54 44.68 42.53 42.35 42.47 31.78 35.43

1464.65 1535.69 1568.69 2020.10 1933.06 1846.03 4891.51 4918.48 6428.41 6626.51 8559.62 8909.38 8759.22 8759.22 8759.22 11723.79 13010.46

1464.65 1535.69 1551.67 2020.10 1933.06 1846.03 4933.52 4960.48 6428.42 6626.51 8559.61 8909.41 8759.24 9415.47 9706.57 11721.79 13006.38

P01034 P01034 P02652

cystatin C cystatin C apolipoprotein A II

35.31 35.31 44.82

13338.60 13338.60 8702.44

13334.61 13350.62 8804.40

P02652

apolipoprotein (truncated) apolipoprotein truncated) apolipoprotein apolipoprotein

A II homodimer

45.21

17146.74

A II heterodimer (entire/

45.15

A II homodimer (entire) AI

45.15 44.10

P02652 P02652 P02647 a

name

PTMsc

Mr (theor. + PTMs)b

des-Ala N-terminal pyroglutamic acid at N-terminal des-Ser N-terminal des-Ser-Ser N-terminal N-terminal acetylation N-terminal acetylation des-Thr-Pro N-terminal

1551.66

4933.52 4960.49

9415.45 9706.54 11721.77 13006.43

17112.79

O-glycosylation O-glycosylation disulfide bond 2 disulfide bonds des-Ser-Ser-Pro-Gly Nterminal 2 disulfide bonds 2 disulfide bonds oxidation Met14 pyroglutamic acid at N-terminal cystenylation 2× pyroglutamic acid at N-terminal

17274.80

17240.70

2× pyroglutamic acid at N-terminal

17240.75

17402.86 28061.47

17368.82 28061.62

2× pyroglutamic acid at N-terminal

17368.80

13334.57 13350.56 8804.41 17112.69

Retention time. bMonoisotopic Mr. cPost-translational modifications.

false-positive identifications among all identifications found and filtering the results for high-confidence identified peptide. The strict target false discovery rate (FDR) value was set to 0.01, while the relaxed value was set to 0.05.

The MS acquisition parameters were the same used for the high-resolution HPLC−MS top-down analysis. MS Data Analysis

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High-resolution MS and MS2 data were elaborated both manually, using the HPLC−MS apparatus management software (Xcalibur 2.0.7 SP1, Thermo Fisher Scientific), and by means of Protein Discoverer 1.2.0 software (2010, Thermo Fisher Scientific) based on SEQUEST cluster as search engine (University of Washington, Seattle, WA, licensed to Thermo Electron, San Jose, CA) against the Swiss-Prot human proteome (Homosapiens_isoform_13022013.fasta, human proteins database, released February 2013). The setting parameters for the top-down/bottom-up data were the following: retention time window 0−61 min; minimum precursor mass 350 Da; maximum precursor mass 10 000 Da; total intensity threshold 0.0; minimum peak count 5; signal to noise (S/N) threshold 3.0; precursor mass tolerance 10.0 ppm; fragment mass tolerance 0.8 Da; use average precursor mass False; use average fragment mass False; maximum retention time difference 0.5 min. Data analysis of bottom-up files was processed with phosphorylation (+79.966 Da) on serine, threonine, and tyrosine; oxidation (+15.995 Da) on methionine; and deamidation (+0.984 Da) on asparagine and glutamine residues as dynamic modification, while carbamidomethylation (+57.021 Da) on cystein residues was set as a static modification. In addition, trypsin enzyme was set. For top-down analysis, no modifications were selected. Protein and peptide identification was carried out using a search against a decoy database that estimates the number of

RESULTS AND DISCUSSION The aim of this work was the identification and characterization of the protein and peptide content of pilocytic astrocytoma intracystic fluid by means of LC−MS using an integrated platform based on both top-down and bottom-up proteomic strategies. The samples were analyzed after a rapid sample pretreatment procedure, already optimized for proteomic analysis of chraniopharyngioma tumor intracystic fluid,10,11 briefly consisting of most abundant protein depletion by ACN under acidic conditions, followed by chloroform liquid/liquid extraction. Intracystic Fluid Top-Down Proteomic Analysis

Figure 1 shows the LC−MS profile obtained from the analysis of an undigested intracystic fluid extract. All analyzed intracystic fluids showed very similar LC−MS profiles. As can be observed in Figure 1A, the chromatogram was mainly characterized by the prevalence of intense peaks in the elution window within 35−47 min time range and of minor peaks at shorter elution times (Figure 1B). From the analysis of the full-scan MS spectra recorded along the chromatogram, a list of deconvoluted molecular masses of proteins and peptides present in the sample was obtained (data not shown). By means of manual inspection of the MS2 spectra of the most abundant multiply charged ions, it was possible to characterize 4597



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Figure 2. Extracted ion current (EIC) plots of entire and N-terminal Ala truncated Fibrinopeptide A in extracted Pilocytic Astrocytoma pediatric brain tumor intracystic fluid analyzed by HPLC-ESI-LTQ-Orbitrap-MS top-down approach. The lower panel reports the deconvoluted ESI full-scan MS spectrum relative of the entire and truncated fibrinopeptide A coeluting peaks. (For detailed operating conditions, see the Experimental Section.)

us to sequence both of the proteoforms and to establish the loss of N-terminal alanine in the truncated form. The experimental mass of fibrinopeptide B showed a difference of −17.02 Da from the theoretical value caused by the cyclization of the Nterminal glutamine residue in pyroglutamic acid. In the recent years, the presence of both fibrinopeptide A and its truncated form was correlated to different types of cancer. Fibrinopeptide A was identified among a panel of biomarkers as the most relevant in diagnosing urothelial carcinoma in urine samples by capillary electrophoresis mass spectrometry (CE−MS) analysis.12 Significantly higher levels of a fibrinopeptide A fragment were found in serum of both gastric cancer patients and high cancer risk individuals with respect to healthy controls, ascribing to this peptide a potential role of diagnostic or predictive disease biomarker.13 As determined by MALDI-MS/MS, the fragment had an m/z value of 1465.64 and was identified as a partial sequence of fibrinopeptide A. This m/z value corresponds to the molecular mass of the N-terminal alanine truncated fibrinopeptide proteoform identified in Table 1. Zhang et al.14 recently

the entire or a part of the sequence of some proteins/peptides. Table 1 reports the list of the proteins and peptides identified, commonly detected in the analyzed pilocytic astrocytoma intracystic fluid samples, together with the relative experimental and theoretical monoisotopic molecular masses (Mr), retention times, Uniprot accession numbers, and identified PTMs. The comparison of the theoretical and experimental MS2 spectra permitted us to confirm the identity of the characterized proteins and peptides together with their hypothesized PTMs. As it can be observed in Table 1, among the identified species, many were interesting bioactive peptides of Mr ranging from 1400 to 4900, such as fibrinopeptide A and its N-terminal truncated form, fibrinopeptide B, complement C3f fragment, and thymosin β4 and β10. All of these peptides were eluting in the 19−22 min retention time window. Figure 2 shows the extracted ion current (EIC) plots of fibrinopeptide A and its N-terminal truncated form together with their deconvoluted full-scan MS spectra in which the presence of two molecular masses differing for an Ala residue (71.03 Da) is evident. The analysis of the MS2 spectra allowed 4598



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Figure 3. Extracted ion current (EIC) plots of Cystatin C in extracted Pilocytic Astrocytoma pediatric brain tumor intracystic fluid analyzed by HPLC-ESI-LTQ-Orbitrap-MS top-down approach. The lower panel reports the deconvoluted ESI full-scan MS spectrum of Cystatin C peak. (For detailed operating conditions, see the Experimental Section.)

confirmed the role of N-terminal alanine truncated fibrinopeptide A as a biomarker of gastric cancer capable of discriminating the presence of lymph-node metastases. The fibrinopeptide A with different PTMs was also identified in serum of patients affected by hepatocellular carcinoma,15 ovarian cancer,16 and metastatic thyroid carcinoma.17 A very recent paper reporting the proteomic analysis of degradome in serum samples of colorectal and prostate cancer patients showed the increase in different fibrinopeptide A fragments in relation to clotting time rather than to the diseases.18 Complement C3f fragment is a heptadeca peptide generated by the catabolic degradation of complement C3b.19 Few articles in literature related complement C3f to cancer diseases. In addition to the entire complement C3f peptide, we identified two C-terminal truncated forms corresponding to the loss of one (monoisotopic Mr 1933.06) and two (monoisotopic Mr 1846.03) N-terminal serine residues. Profumo et al.20 identified both complement C3f and some of its fragments in serum of patients affected by breast’s benign cystic disease. In particular, increased level of these peptides and especially of entire C3f exhibited a potential employment in predicting cancer outcome even 20 years later. Additionally, complement C3f fragments

showed higher serum levels in association with thyroid carcinoma17 and were included in a panel of cancer signature peptides in a paper studying breast, bladder, and prostate cancers.21 Differently, in serum of patients affected by colorectal cancer22 and hepatocellular carcinoma,23 the levels of different C3f peptide fragments (N- or C-terminal of both terminals truncated), including the Mr 1934.93, were found to decrease. Two fragments of complement C3f peptide, the des-Arg and its derivative, differing only for one amino acid residue loss in the sequence, were significantly increased in acute leukemia and decreased in association to disease remission grade, therefore resulting in potential biomarkers of treatment and prognosis.24 Similarly to craniopharyngioma intracystic fluid,10 both thymosins β4 and β10 peptides were identified in pilocytic astrocytoma intracystic fluid, probably as products of tumor cell secretion into the extracellular environment. Thymosin β4 and β10 are G-actin sequestering peptides involved in multiple functions such as modulation of cell motility, tissue repair, and anti-inflammatory response.25 In relation to tumors, thymosin β4 shows an antiapoptotic role, hence enhancing tumor cells proliferation and metastasis formation26 by the activation of angiogenesis and cellular migration processes.27 High levels of 4599



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Figure 4. Identification of apolipoprotein A II in extracted Pilocytic Astrocytoma pediatric brain tumor intracystic fluid by HPLC-ESI-LTQOrbitrap-MS top-down approach. The Figure illustrates the chromatographic peaks of the TIC LC−MS profile where apolipoprotein A II was identified (panel A, peaks eluted at 44.84 and 45.15 min) together with the relative ESI full-scan MS spectra (panels B and C, respectively) and their deconvolution (panels D and E). Panel F shows the deconvoluted ESI full-scan MS spectrum of the chromatographic peak eluted at the same retention time of the peaks in panel A of the analyzed pylocitic astrocytoma intracystic fluid after treatment for disulfide bond reduction. (Experimental details are reported in the Experimental Section.)

thymosin β10 have been associated with cancers progression and metastasis,26 but the molecular mechanism involved is still controversial.28

As it can be observed in Table 1, together with several peptides, the intracystic fluid of pilocytic astrocytoma also showed the presence of several proteins: different apolipopro4600



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teins, cystatin C, β-2 microglobulin, and ubiquitin with several and interesting PTMs. Cystatin C, already characterized in our laboratory in saliva samples, 29 eluted at 35.3 min. Figure 3 depicts the corresponding LC−MS EIC profile and the deconvoluted ESI full-scan MS spectrum of Cystatin C (13335.62 [M + H]+, monoisotopic). The experimental Mr of Cystatin C differed from the calculated theoretical value due to the presence of two disulfide bonds in the molecule, as also reported in Uniprot database, and was confirmed by theoretical/experimental MS2 spectra comparison. As resulting from the deconvolution of the ESI full-scan MS spectrum (Figure 3, lower panel), Cystatin C peak showed the coelution of two additional proteins of 13351.62 and 13007.39 m/z ([M + H]+, monoisotopic) differing by +16.00 and −328.23 Da, respectively, from Cystatin C molecular mass. The hypothesis was that the 13351.62 m/z protein was originating from Cystatin C after oxidization of one methionine residue and the 13007.39 m/z (Cystatin C shorter form) protein from a possible truncation at the N- or Cterminal sides. The comparison of the experimental MS2 spectra with all possible theoretical ones containing an oxidized methionine residue allowed identifying the site of the modification at Met14 in the 13351.62 m/z protein. The same procedure was applied to the shorter form of Cystatin C that resulted from the removal of the first four N-terminal amino acid residues (-SSPG missing, N-terminal). These two Cystatin C proteoforms are not reported in Uniprot database. However, in the literature, four different Cystatin C proteoforms have been described: the entire Cystatin C and the socalled slow, intermediate, and fast cystatin C, the last three corresponding to different N-terminal truncated forms.30,31 In particular, the slow and the fast forms are the most common in human fluids. In our samples, we detected the slow cystatin C, but we did not observe the fast form. These two peptides, probably generated by different N-terminal cleavages occurring during cellular inflammatory response,32 differ by the four Nterminal amino acid residues, the slow form (des 1−4) containing the N-terminal tetrapeptide (LysProProArg) with respect to the fast form (des 1−8). The slow form of Cystatin C has been reported to be involved in the modulation of immune defense during inflammation acting on neutrophil behavior, particularly inhibiting their oxygen release and phagocytosis functions. This effect was not shown by the fast form and seemed to be due to the release of the N-terminal tetrapeptide, called “postin”, from the slow proteoform.32 β-2 microglobulin, similarly to Cystatin C, is involved in immune response, and it is part of the HLA (Human Leukocyte Antigen) system. Altered levels of this protein were found in association with several diseases, including some types of cancer, such as multiple myeloma or lymphoma, suggesting a role as CNS inflammatory or tumor marker in CSF.33 Figure 4 illustrates the full-scan MS spectra deconvolution corresponding to the adjacent peaks at retention times of 44.84 and 45.15 min. As it can be observed from Figure 4C, the peak at 45.15 min showed an ESI full-scan MS spectrum characterized by three different multiplycharged ion Gaussian distributions originated by proteins with very similar molecular mass around 17 000 Da (relative spectrum deconvolution in Figure 4E) and probably corresponding to diverse isoforms of the same protein. The MS2 spectrum of the 1580.99 m/z multiplycharged ion (z = +11) allowed us to identify part of the amino acid sequence that Blast software (Expasy data bank) assigned with high probability to Apolipoprotein A II (apo AII).

This protein can exist as monomer or can form dimers by means of disulfide bonds involving Cys6 residue, even though the observed experimental masses did not correspond to the theoretical values of possible dimers: the protein with the highest molecular mass (17369.83 m/z, [M + H]+), showing a −34.23 Da mass difference from the theoretical value of apo AII homodimer, was possibly ascribed to the homodimer with a pyroglutamic acid at both N-terminal Gln residues. The other molecular masses found, namely, the 17 241.71 and the 17 113.80 m/z ([M + H]+), showed a delta mass of 128.12 and 256.03 Da from the theoretical ones, possibly compatible with the presence in the dimer of one or two C-terminal Gln truncated monomers, respectively. Therefore, the identified apo AII dimers resulted from various assortments of diverse monomers: (i) two entire monomers (Apo AII homodimer), (ii) two C-terminal truncated monomers (truncated Apo AII homodimer), and (iii) one entire and one C-terminal truncated monomer (entire/truncated apo AII heterodimer), all presenting N-terminal pyroglutamic acid. The use of high-resolution mass spectrometry allowed discriminating the different dimeric forms and the free monomers. Figure 4B,D shows the full-scan MS spectrum and its relative deconvolution of the peak at 44.84 min showing the presence of apo AII monomers of 8805.41 and 8677.38 m/z ([M + H]+), corresponding to both cysteinylated entire and C-terminal truncated isoforms, respectively. All of these monomers showed a molecular mass difference from the theoretical value compatible with Nterminal pyroglutamic acid formation, already characterized in the corresponding dimers, further increased of +119.00 Da justified by a cysteinylation at Cys6 residue, confirmed by MS2 spectra. As it can be observed in the deconvoluted spectra of Figure 4D,E, the monomers were detectable in both the chromatographic peaks at 44.84 and 45.15 min, resulting in copresence of the relative dimers in the second one. As it can be observed in panels D and E, two other intense molecular masses were additionally detected, 8910.42 (panel D) and 8652.43 (panel E) m/z, that were investigated to establish if correlated to apo AII. The 8652.43 m/z protein was identified as the cysteinylated entire apo AII (8805.41 m/z), also present in panel D, in which the cysteinylated cystein residue was converted to dehydroalanine (DHA) by the successive loss of cysteinylation and SH2. Cystein conversion to DHA occurs at high temperature and was probably an artifact generated during ESI source ionization. In fact, this monomer was not present anymore in the spectrum after disulfide bonds reduction (Figure 4 F). Differently, the 8910.42 m/z was characterized as Apolipoprotein C II by manual sequencing of the deconvoluted MS2 spectrum registered on the 1274.5 m/z (z = +7), therefore adding the detection of another interesting apolipoprotein to the previous apolipoprotein list of the analyzed samples. To further confirm the identification of apo AII monomeric and dimeric proteoforms, we treated selected samples for disulfide bonds reduction by IA alkylating agent. As can be observed in Figure 4F, the deconvoluted ESI full-scan MS spectrum recorded at the same retention time of apo AII dimers showed the presence of four different molecular masses ([M + H]+) corresponding to both the entire (8686.43 m/z) and C-terminal truncated (8558.51 m/z) monomers and to their relative carbamidomethylated derivatives (8743.47 and 8615.43 m/z, respectively). The carbamidomethylation reaction on cysteine residue resulted nonquantitative, giving rise to a mixture of carbamidomethylated and noncarbamidomethylated 4601



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Figure 5. Scheme of the apolipoprotein A II proteoforms identified in extracted astrocytoma pilocytic pediatric brain tumor intracystic fluid by HPLC-ESI-LTQ-Orbitrap-MS top-down approach in which all molecular masses ([M + H]+, monoisotopic) of the monomers and dimers identified in both unreduced and disulfide bond reduced samples are reported. CMC, cysteine residues carbamidomethylation.

entire and truncated monomers. Figure 5 summarizes all apo AII monomeric and dimeric isoforms identified in astrocytoma pylocitic intracystic fluid in both disulfide-bond reduced and unreduced samples. Although the possible formation of apo AII homo- and heterodimers is described in Uniprot database, the presence of pyroglutamic acid at N-terminal glutamine is not reported, and, in addition, the relative molecular masses listed, determined by MALDI-MS, show a delta mass from both the theoretical average and monoisotopic Mr values, which instead exactly matched our experimental findings. Other interesting proteins were identified in the chromatographic window between 41 and 43 min. In particular, the MS spectrum deconvolution of the peak eluting at 42.41 min and the sequencing of the relative MS2 spectra allowed the characterization of apolipoprotein C III and its probable glycosylated isoforms. The sequencing of the primary structure of glycosylated proteoforms was not allowed due to the preferential fragmentation of polysaccharide chain over the protein/peptide backbone. However, high-resolution mass spectrometry permitted to possibly assign the delta mass observed between the different protein species to diverse glycoforms of the same protein (Figure 6). The 8963.33 m/z proteoform showed a delta mass of +203.09 Da from apolipoprotein C III (8760.25 m/z, [M + H]+), possibly corresponding to acetylgalactosamine (GalNAc) or acetylglucosamine (GlcNAc) N-glycosylation, whereas the 9125.38 m/z proteoform showed a further difference of +162.05 Da corresponding to a subsequent addition of an hexose unit in the glycosidic chain. The 9416.48 and 9707.58 m/z glycoforms differed from the 9125.38 m/z for the presence of one and two residues of sialic acid, respectively. Our experimental findings were confirmed by the report in Uniprot database of

apolipoprotein C III PTM glycosylations according to the identified glycosylated chain structures. The elaboration of the top-down LC−MS/MS analysis by the bioinformatic software “Proteome Discover 1.2.0” confirmed our experimental findings and contributed with new potential identifications. The results confirmed the presence of Apolipoprotein C I, Fibrinopeptide A, and complement C3f fragment and additionally identified Apolipoprotein J, or clusterin, and Apolipoprotein A I (data not shown) in some samples. The presence of Apolipoprotein A I was therefore manually confirmed in all samples analyzed and therefore included in Table 1 as a commonly present protein. All analyzed pilocytic astrocytoma intracystic fluids resulted strongly characterized by the presence of different proteins belonging to the apolipoprotein family. Together with their entire forms, different isoforms were additionally identified containing diverse PTMs including truncated forms, homo- and heterodimerization, glycosylation, and oxidations. Apolipoproteins play a very important role in lipid transport and metabolism, especially in the brain, the organ at higher lipid content and isolated by the blood−brain barrier (BBB). Some experimental evidence showed that they also carry out other important functions essential to the functionality of a healthy organism, and interesting correlations to different diseases, including tumors, have been in addition outlined.34 Apolipoprotein A I was proposed as a serum biomarker of hepatocellular carcinoma35 and, together with apolipoprotein A II, as a urinary biomarker of bladder cancer.36 Apolipoprotein A I function in the brain is still unclear. It was identified in CSF and brain tissues, and its metabolism and concentration in CNS seems to be independent from other compartments of the organism.34 Apolipoprotein A II has been proposed as indicator 4602



Article

Figure 6. Deconvoluted ESI full-scan MS spectrum relative to apolipoprotein C III peak in extracted pylocitic astrocytoma intracystic fluid analyzed by HPLC-ESI-LTQ-Orbitrap-MS top-down approach. (For detailed operating conditions, see the Experimental Section.)

of BBB damage in relation to pediatric brain tumor diseases37 and resulted overexpressed in serum of prostate cancer patients.38 Apolipoprotein C I is expressed in the brain, even if the protein and its concentration decreases with aging,34 and was correlated to proinflammatory response and survival of pancreatic tumor cells, preventing apoptosis.39 Similarly, Apolipoprotein C II was recently identified as a prognostic biomarker in pancreatic cancer40 and in advanced cervical cancer after chemoradiation therapy.41 Apolipoprotein C III, the most abundant within C-type-apolipoproteins, can be differently glycosylated generating diverse isoforms,42 which we were able to characterize by high-resolution mass spectrometry top-down approach, as above-reported (Figure 6). Apolipoprotein J is expressed in many parts of the brain and shows increased levels in neurodegenerative pathologies, ischemia, and tumors, probably with a protective role.43 The software for the bioinformatic analysis is limited to the characterization of entire proteins or protein fragments within 10 000 Da, therefore hindering the presence of higher molecular mass proteins present in the samples. The identification of proteins with Mr > 10 000 was accomplished by manual identification or application of a bottom-up proteomic strategy.

In addition to serum albumin, commonly and extensively present in all of the samples, several other proteins of interest for the pathology under examination have been identified. Apolipoproteins A I, A II, and C II, strongly characterizing the tumor fluid, were identified also by the bottom-up approach, confirming the top-down proteomics detections. In addition, the bottom-up analysis recognized the presence of a new apolipoprotein, the Apolipoprotein D with monoisotopic Mr of 19 290.67 together with other proteins, some of them ubiquitarily found in the analyzed samples or in the majority of them, such as the isoform 2 of vitamin Dbinding protein, the alpha-1-acid glycoproteins 1 and 2, and the isoform 2 of TRAF3-interacting protein 1. Apolipoprotein D expression was differently correlated to CNS astrocytoma and medulloblastoma tumors, resulting in an inverse measure of proliferation and a diagnosis biomarker.44 It is noteworthy to underline that by the application of the bottom-up strategy, some information about isoforms and PTMs could be lost, as the connectivity within the peptide chains is missed in consequence of the enzymatic cleavage.

■

CONCLUSIONS The first proteomic characterization of pilocytic astrocytoma intracystic fluid by the application of LC−MS top-down/ bottom-up integrated approach is shown here. The application of both the proteomic strategies resulted complementary, allowing us to reciprocally confirm and combine the experimental data findings, resulting in a successful integrated proteomic platform. By means of top-down approach,

Intracystic Fluid Bottom-Up Proteomic Analysis

The bottom-up proteomic approach was applied to all analyzed samples of pylocitic astrocytoma intracystic fluid. The elaboration of the LC−MS/MS data by Proteome Discover 1.2.0 software produced the list of proteins reported in Table 2. 4603



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Table 2. List of Proteins Identified in Pilocytic Astrocytoma Intracystic Fluid from Data Elaboration of Bottom-Up Proteomic LC−MS Analysis of Different Patient Samples patients accession P02768 P02652 P02787 P01834 P02647 P68871 P05090 P02774-2 P02655 P00738 P02763 P69905 P19652 O76096 P04217 Q9C0E2 P12821-2 Q8TDR0-2 P20936-2

description serum albumin OS=Homo sapiens GN=ALB PE=1 SV=2 - [ALBU_HUMAN] apolipoprotein A-II OS=Homo sapiens GN=APOA2 PE=1 SV=1 [APOA2_HUMAN] serotransferrin OS=Homo sapiens GN=TF PE=1 SV=3 - [TRFE_HUMAN] Ig kappa chain C region OS=Homo sapiens GN=IGKC PE=1 SV=1 [IGKC_HUMAN] apolipoprotein A-I OS=Homo sapiens GN=APOA1 PE=1 SV=1 [APOA1_HUMAN] hemoglobin subunit beta OS=Homo sapiens GN=HBB PE=1 SV=2 [HBB_HUMAN] apolipoprotein D OS=Homo sapiens GN=APOD PE=1 SV=1 - [APOD_HUMAN] isoform 2 of vitamin D-binding protein OS=Homo sapiens GN=GC [VTDB_HUMAN] Apolipoprotein C−II OS=Homo sapiens GN=APOC2 PE=1 SV=1 [APOC2_HUMAN] haptoglobin OS=Homo sapiens GN=HP PE=1 SV=1 - [HPT_HUMAN] alpha-1-acid glycoprotein 1 OS=Homo sapiens GN=ORM1 PE=1 SV=1 [A1AG1_HUMAN] hemoglobin subunit alpha OS=Homo sapiens GN=HBA1 PE=1 SV=2 [HBA_HUMAN] alpha-1-acid glycoprotein 2 OS=Homo sapiens GN=ORM2 PE=1 SV=2 [A1AG2_HUMAN] cystatin-F OS=Homo sapiens GN=CST7 PE=1 SV=1 - [CYTF_HUMAN] alpha-1B-glycoprotein OS=Homo sapiens GN=A1BG PE=1 SV=4 [A1BG_HUMAN] exportin-4 OS=Homo sapiens GN=XPO4 PE=1 SV=2 - [XPO4_HUMAN] isoform somatic-2 of angiotensin-converting enzyme OS=Homo sapiens GN=ACE [ACE_HUMAN] isoform 2 of TRAF3-interacting protein 1 OS=Homo sapiens GN=TRAF3IP1 [MIPT3_HUMAN] isoform 2 of Ras GTPase-activating protein 1 OS=Homo sapiens GN=RASA1 [RASA1_HUMAN]

interesting protein PTMs were characterized that could have an important role in the comprehension of disease onset and development, such as truncated, oxidized, and glycosylated proteoforms, while the bottom-up approach allowed us to recognize the presence of high-molecular-mass proteins and to widen the proteome characterization. Of particular interest was the identification of several proteins belonging to the apolipoprotein family presenting different PTMs, including the presence of dimeric forms, and the characterization of truncated cystatin C, missing the first four N-terminal amino acid residues, reported to strongly modulate the immune defense mechanisms. The identified proteins are involved in different biological processes such as regulation of inflammatory response, immune response, lipid removal, cell growth, development and differentiation, protein turnover and degradation (ubiquitin), and tumor cell mechanisms of proliferation. Our data provide new insights into the characterization of pilocytic astrocytoma tumor fluid and could be of help in the understanding the genesis and development of the cyst together with the onset and progression of the disease, additionally resulting in support for a possible updating of the available bioinformatic databases by high-resolution molecular mass spectrometry sequencing. Further experiments will be devoted to assess the potential specificity of the identified proteins and peptides inside pilocytic astrocytoma intracystic fluid by comparing the proteomic results obtained from other cerebral cystic fluids

Σ coverage

Mw [kDa]

∑# unique peptide

1

2

3

4

89.00% 49.00%

69.3 11.2

90 5

×

× ×

× ×

× ×

41.83% 33.02%

77 11.6

15 2

×

× ×

31.46%

30.8

6

29.25%

16

4

24.34% 24.15%

21.3 39.5

3 5

23.76%

11.3

1

12.56% 12.44%

45.2 23.5

3 2

10.56%

15.2

1

4.98%

23.6

1

4.83% 4.04%

16.4 54.2

1 1

3.91% 2.79%

130.1 131.6

1 1

× ×

2.72%

71.5

1

×

2.30%

100.3

1

× ×

× ×

×

×

× × ×

×

× ×

×

×

× ×

×

×

× × × × ×

× ×

related to both oncological and nononcological pediatric diseases, possibly disclosing potential candidate disease biomarkers.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions #

I.I. and F.I. contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We acknowledge the financial support of Italian National Research Council (CNR), Catholic University of Rome and Cagliari University according to their programs of scientific diffusion.

■

REFERENCES

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Proteomic Study of Pilocytic Astrocytoma Pediatric Brain Tumor

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