Explorative Study of the Protein Composition of Amniotic Fluid by

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Explorative Study of the Protein Composition of Amniotic Fluid by Liquid Chromatography Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Stefan Nilsson,†,| Margareta Ramstro1 m,†,| Magnus Palmblad,‡,|,# Ove Axelsson,§ and Jonas Bergquist*,† Department of Analytical Chemistry, Uppsala University, Box 599, SE-751 24 Uppsala, Sweden, The Ångstro¨m Laboratory, Division of Ion Physics, Uppsala University, Box 534, SE-751 21 Uppsala, Sweden, and Department of Women’s and Children’s Health, Obstetrics and Gynecology, Uppsala University, Akademiska sjukhuset, SE-751 85 Uppsala, Sweden Received February 13, 2004

To explore the suitability of FTICR mass spectrometry for the analysis of the protein composition of amniotic fluid (AF), an AF sample from 15 weeks gestation from a healthy 36-year-old woman was tryptically digested, with or without prior serum albumin removal. The tryptic peptides were separated by gradient capillary liquid chromatography (LC) followed by electrospray ionization (ESI) and mass spectrometric detection with a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR). The obtained data underwent computer-aided mathematical and statistical evaluation to extract significant tryptic peptide patterns from human proteins. Forty-three proteins were putatively identified; among them were known protein constituents of amniotic fluid, but also many that not have been detected before. The removal of serum albumin prior to tryptic digestion reduced ion suppression from abundant HSA fragments. The protein analysis of amniotic fluid by albumin removal, tryptic digestion and LC/FT-ICR-MS analysis was found to be a straightforward technique. Keywords: amniotic fluid ‚ data handling ‚ depletion ‚ LC/ESI-FT-ICR-MS ‚ proteomics

Introduction Accurate knowledge of the mass of an unknown molecule may reveal its identity. Among several ways for molecular mass determination, Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS)1 provides extraordinary mass resolution and mass accuracy. For the analysis of complex liquid samples, the most common instrumental combination is liquid chromatography (LC) hyphenated to MS through an electrospray ionization2 source (LC/ESI-MS). Several recent papers3-9 describe the use of ESI-FT-ICR-MS for the computer-aided identification of proteins in extremely complex samples derived from different human body fluids, such as cerebrospinal fluid, plasma, urine and saliva. In particular, the combination of LC/ ESI-FT-ICR-MS has proved to be advantageous for such analysis.4,7-9 In this paper, the combination is tried on an amniotic fluid (AF) sample to reveal its potency for this type of sample. To our knowledge, this is the first attempt to use an MS-based proteomic approach on AF. * To whom correspondence should be addressed. E-mail: Jonas. [email protected]. Fax: +46 (0)18 471 3692. † Department of Analytical Chemistry, Uppsala University. ‡ The Ångstro¨m Laboratory, Division of Ion Physics, Uppsala University. § Department of Women’s and Children’s Health, Obstetrics and Gynecology, Uppsala University. | These authors contributed equally to this work. # Present affiliation: Center for Accelerator Mass Spectrometry, L-397, Lawrence Livermore National Laboratory, Livermore, CA 94550.

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Amniotic fluid surrounds the fetus in the amniotic cavity, which from gestational week 10-12 is in close contact with the chorion and thus the uterine wall. At 15 weeks gestation, the volume is about 200 mL, but with substantial individual variations. At that time, fetal urine and fetal alveolar fluid are the main contributors to AF whereas fetal swallowing and transport through the amniotic-chorionic interface removes fluid.10 AF contains cells of fetal origin as well as other fetal products e.g. proteins.11 This forms the basis for prenatal diagnosis through analysis of amniotic fluid. Knowledge of the protein composition of the AF is then of outmost importance as every known AF protein is a potential marker for fetus health, alone or in combination with other information. The purpose of this study was to investigate whether LC/ESI-FT-ICR-MS could be useful for the search of amniotic fluid proteins.

Experimental Section Samples. The sample was taken at 15 weeks gestation from a healthy 36-year-old woman, who has opted for an amniocentesis due to age. A transabdominal technique was used and 13 mL of clear amniotic fluid was withdrawn, of which 3 mL was designated for the study. The pregnancy went on without complications and ended with a delivery of a healthy female infant at term. The chairman of the Ethics Committee at the Faculty of Medicine approved the study. 10.1021/pr0499545 CCC: $27.50

 2004 American Chemical Society

Protein Composition of Amniotic Fluid

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Figure 1. Flowchart of the data analysis. The score is arbitrary but proportional to the likelihood ratios and used to compare with random sequences to estimate statistical confidence in protein identifications.

Sample Handling. Twenty minutes after the withdrawal of sample from the amniotic cavity, the sample had been divided into aliquots of 100 µL in siliconized tubes (Costar, Corning Inc., New York, USA) and stored in a -80 °C freezer. The total protein content of the amniotic fluid was determined, using the bicinchoninic acid protein assay reagent (Pierce Chemical Company, Rockford, USA), to be 3.3 g/L, which is in the expected range.12 Aliquots of 50 µL amniotic fluid, corresponding to 165 µg total protein each were used for further investigation. Two methods for sample preparation were tested and compared. In the first approach, human serum albumin (HSA), being the dominating protein in the fluid, was removed from the sample before the remaining proteins were tryptically digested and analyzed by LC/FTICRMS. The method has previously proven to be advantageous when studying human plasma.13 In the second approach, the proteins in AF were directly cleaved by trypsin prior to analysis. Hence, two different samples were prepared. In Sample 1, HSA was removed using a HSA depletion kit (Millipore Corporation, Bedford, MA). The removal of HSA was performed according to the standard protocol (www.Millipore.com). Sample 2 was kept intact. Both samples were vacuum centrifuged to dryness, using a Speedvac system ISS110 (Savant Holbrook, New York, USA). The pellets were dissolved in 100 µL of 8 M urea, 0.4 M NH4CO3, after which 10 µL of 45 mM dithiothreitol was added. The samples were kept at 50 °C for 15 min. Ten µL of 100 mM iodoacetamide was added, followed by incubation for 15 min at room temperature in darkness. Six µg of trypsin from bovine pancreas (1 418 475), Boehringer Mannheim GmbH, Mannheim, Germany), dissolved in 130 µL water were added to each sample, and the tryptic digestion took place overnight at 37 °C. The tryptic digests were desalted on ZipTip C18 tips (Millipore Corporation, Bedford, USA). This procedure is described in detail elsewhere.8 After desalting, the samples were again vacuum centrifuged to dryness.

Liquid Chromatography and Mass Spectrometry. The dried samples were dissolved in 10 µL of buffer A, and injected on the LC-column in a six-port injector valve (Valco Instruments Co. Inc., Schenkon, Switzerland). The peptides were separated on a 10 cm long packed capillary C18-column of an internal diameter of 200 µm. The packing material was L 5 µm ODSAQ (YMC Europe, Schermbeck, Germany). A mobile phase gradient was delivered by two JASCO 1580 HPLC-pumps and a JASCO HG-1580-32 Dynamic Mixer (JASCO, Tokyo, Japan) at a flow rate of 1 µL/min over the column. An isocratic step using 100% A for 10 min was followed by a gradient 0-50% B in 64 min followed by 50-100% B in 5 min where mobile phase A was acetonitrile (ACN):H2O:HAc (5:94.5:0.5 v:v:v), and mobile phase B ACN:H2O:HAc (94.5:5:0.5 v:v:v). The peptides passed a UV-detector (JASCO UV-1575) before they were electrosprayed on-line to a Bruker Daltonics BioAPEX-94e 9.4 T Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA)14 using a Black Dust15 (polyimide-graphite) sheathless electrospray emitter, i.d. 50 µm. Data Handling. Data were collected on a computer running Xmass. Xmass was started 24 min after injection of the sample in the LC-system. In total, 256 mass spectra of 256 K data points were created in each experiment. Each spectrum was collected during 13 s. Internal mass calibration was performed between m/z 671 and m/z 1706 using 32 abundant HSA and transferrin (proteins known for being abundant in AF16) peptides distributed throughout the chromatographic run. The mean mass measurement error was around 5 ppm for the 32 calibration peptides and less than half for the 16 peptides between m/z 686 and m/z 988, which is also the interval where most peptides are found. The complete 2D data sets from Xmass were submitted to software written in-house. A flowchart of the data treatment is shown in Figure 1. The computer program compared measured peptide masses, sequence maps, and retention times with those predicted for peptides generated by digesting human protein Journal of Proteome Research • Vol. 3, No. 4, 2004 885

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To decrease the risk for false positive hits, a subset of database proteins were selected according to size (2-85 kDa) and pI (113), resulting in 23 796 sequences. The chromatographic retention time predictor was trained using a method described by Palmblad et al.,4 i.e., by 21-dimensional least-squares fitting to measured retention data for a large number of peptides. In this case, 175 peptides from four proteins known to be present in AF, and putatively identified by accurate mass and high relative intensity in the mass spectra from the experiments was used as training set. An estimate of the accuracy of the predictor when applied on the data from Sample 1 is shown in Figure 2. Out of the peptides in the training set, 93% (163 peptides) were predicted within 20% relative error and 75% or 131 of the 165 peptides could be predicted with less than 10% relative error. All software for data analysis was written in-house in ANSI C, compiled by gcc 3.3.1 and run on a 2.4 GHz/512 MB RAM Pentium 4 personal computer. The database searching takes just over a minute for the datasets generated by the LC/FTICR-MS analysis.

Results and Discussion Figure 2. Accuracy of the chromatographic retention time predictor when trained using 175 peptides from the four proteins listed in the text putatively identified by accurate mass and high relative intensity in the mass spectra. The dotted lines indicate 20% deviations.

sequences in silico, using likelihood ratios, as previously described by Palmblad et al.4,8,17,18 The Swiss-Prot/TrEMBL human protein database (totally 27 963 sequences) was searched.

An example of the LC-MS raw data is shown in Figure 3. For Sample 1, using a variable background threshold, 49 241 mass peaks were found. These were subsequently deconvoluted to 2185 unique peptide masses, which were matched against in silico digested proteins as described in the Experimental Section. In the first round, proteins were identified based on accurate mass alone. Four human sequences scored higher than the highest scoring archaeal negative control (these were consistently the four highest scoring sequences in repeated experiments, Swiss-Prot/TrEMBL identification number in

Figure 3. Transformed raw data from the LC/ESI-FT-ICR-MS for Sample 1. The inset corresponds to the marked region and illustrates complexity of the dataset. 886

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Protein Composition of Amniotic Fluid

Table 1. Top Scoring Human Proteins in Amniotic Fluid for Sample 1, Where Albumin Was Depleted before the Tryptic Digestion (see Experimental Section) score

protein name (Swiss-Prot)

SPTr ID

comment

4.8 × 103 3.8 × 103 1.3 × 103 5.8 × 102 3.7 × 102 2.0 × 102 1.9 × 102 1.9 × 102 1.9 × 102 1.9 × 102 1.7 × 102 1.5 × 102 1.5 × 102 1.3 × 102 1.3 × 102 1.1 × 102 1.1 × 102 1.0 × 102 9.9 × 101 9.3 × 101 9.2 × 101 9.2 × 101 9.2 × 101 9.0 × 101 9.0 × 101 8.9 × 101 8.7 × 101 8.7 × 101 8.3 × 101 8.2 × 101 7.7 × 101 7.6 × 101 7.5 × 101 7.4 × 101 7.4 × 101 7.0 × 101 7.0 × 101 6.9 × 101 6.9 × 101 6.9 × 101 6.9 × 101 6.7 × 101 6.5 × 101

serum albumin transferrin R-1-antitrypsin R-fetoprotein vitamin D-binding protein hypothetical protein hypothetical protein DNA-directed RNA polymerases III 80 kDa polypeptide DNA-directed RNA polymerases III 80 kDa polypeptide DNA-directed RNA polymerases III 80 kDa polypeptide zinc finger protein 221 protein p84 P47 protein zinc finger protein AMBP protein hypothetical protein KRAB zinc finger protein hypothetical protein RNA-binding region containing protein 2 hypothetical protein Ig κ chain C region U3 small nucleolar ribonucleoprotein protein MPP10 similar to egl hypothetical protein formin-like protein paired box protein Pax-3 hypothetical protein similar to RIKEN cDNA A930031F18 gene calpain 6 hypothetical protein FLJ00239 protein fragment G4 protein hypothetical protein hypothetical protein hypothetical protein PRO2275 hypothetical protein B lymphocyte adapter protein BAM32 zinc-R-2-glycoprotein adaptor protein DAPP1 R-2-glycoprotein 1, zinc keratin, type I cytoskeletal 14 pinin

P02768 P02787 P01009 P02771 P02774 Q96JN0 Q8N3L6 Q9P276 Q9BWF7 Q9NVU0 Q9UK13 Q15219 Q9UI06 Q9NR94 P02760 Q96JV2 Q96JC4 Q9H809 Q14498 Q8NAG0 P01834 O00566 Q8NHP7 Q8N9K9 O95466 P23760 Q8N800 Q9BT92 Q9Y6Q1 Q96GE5 Q8TEF8 Q9UMP7 Q9Y228 Q8WWC4 Q8NAV5 Q9P173 Q96PX6 Q9UHF2 P25311 Q9UN19 Q8N4N0 P02533 Q99738

colloidal protein; unspecific binding iron transport serine protease inhibitor colloidal, unspecific binding; immunosuppressive21 vit D and actin monomer binding

Table 2. Top Scoring Archaeal Sequences score

6.4 ×

101

6.3 × 101 5.7 × 101 5.6 × 101 4.9 × 101

protein name (Swiss-Prot)

putative D-3-phosphoglycerate dehydrogenase putative acetyl-CoA synthetase carbamoyl-phosphate synthase large chain, N-terminal section DTDP-glucose 4,6-dehydratase tRNA nucleotidyltransferase

SPTr ID

Q974F8 Q96Z08 Q8TWX0 Q9UXJ4 Q97Z92

brackets): serum albumin (P02768), transferrin (P02787), R-1antitrypsin (P01009), and R-fetoprotein (P02771). When the nonrandomness of proteolytic peptide maps was incorporated, 28 human proteins scored higher than the highest scoring negative control. When factoring in the nonrandomness of predicted peptides retention times, 43 human proteins (Table 1) scored higher than the highest scoring archaeal protein (Table 2). Using retention time prediction but not nonrandomness, 15 human sequences scored higher than the highest scoring archaeal protein. The likelihood scores of the negative controls typically have a smooth distribution with few outliers. Since there were more

DNA transcription DNA transcription DNA transcription transcription factor? transcription complex protein proteolytic inhibitor, colloidal protein

transcriptional coactivator part of immunoglobulin RNA processing

probable transcription factor placental protein

stimulates lipid catabolism antigen presentation intermediate filament placental protein

human proteins than archaeal proteins in the mass filtered database, up to 5-6 false positives could be expected among the proteins scoring higher than the highest scoring archaeal sequence. The statistical significance of the highest scoring human sequences is hard to estimate as there are no random hits with such high scores and due to the bias introduced when internally calibrating mass spectra and internally training the retention time predictor using peptides from the top scoring proteins. However, the most highly scoring sequences are all known to be abundant in amniotic fluid.16 In addition, even the lowest scoring protein in Table 1, pinin, has been found in the placenta.19 In the sample prior to HSA removal, Sample 2, 50 570 mass spectral peaks could be reduced to 2036 unique masses. After internal calibration and training of the predictor as described above, 20 human proteins scored higher than the highest scoring negative control, HSA being by far the highest scoring sequence (Table 3). When comparing the signal of the peptides between the two fractions, it seems that HSA causes significant ion suppression and space charge effects due to its high abundance in the AF. Presumably, there is much less HSA after HSA has been affinity-captured and removed from the sample. However, the total intensity of all HSA peptides (the same Journal of Proteome Research • Vol. 3, No. 4, 2004 887

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Table 3. Top Scoring Human Sequences in Amniotic Fluid for Sample 2, Where No Albumin Depletions Were Performed (see Experimental Section) score

protein name (Swiss-Prot)

SPTr ID

comment

2.32 × 103 1.55 × 102 1.46 × 102 1.21 × 102 1.00 × 102 9.73 × 101 8.96 × 101 7.45 × 101 6.71 × 101 5.16 × 101 4.79 × 101 4.61 × 101 3.63 × 101 3.58 × 101 3.48 × 101 3.47 × 101 3.32 × 101 3.32 × 101 3.27 × 101 3.10 × 101

serum albumin transferrin R-fetoprotein unlikely hyphotetical protein zinc finger protein 189 Ig γ-1 chain C region prospero-related homeobox 1 homeobox prospero-like protein PROX1 R-1-antitrypsin hypothetical protein BA74P14.2 kelch-like protein 4 syntaxin 11 type XIII collagen sir2-related protein type 7 eosinophil peroxidase R-1 type XIII collagen hypothetical protein SCO2 protein homolog, mitochondrial hematopoietic PBX-interacting protein

P02768 P02787 P02771 Q8TC77 O75820 P01857 Q8TB91 Q92786 P01009 Q9H955 Q96QW3 Q9C0H6 O75558 Q9NQ52 Q9NRC8 P11678 Q99228 Q9NW10 O43819 Q96AQ6

colloidal protein; unspecific binding iron transport colloidal, unspecific binding; immunosuppressive21

numbers were detected in both samples) decreased only by ∼20%. The number and total intensity of peptides putatively corresponding to the other abundant proteins increased markedly after HSA removal. The total intensity of all peptides differed by 1.3% between the two runs. The trend is the same, albeit less emphasized, also when normalizing to the number of peptides found for each protein or looking at the exact same peptides. However, we cannot conclude that removal of HSA is solely beneficial for this type of experiment, since losses of other proteins bound to HSA may occur. Among the identified proteins from both samples, some sequences are very similar, e.g., the hypothetical proteins Q96JNO and Q8N3L6 (93% identical, differing only in Nterminal sequence and three gaps in Q96JNO). The 15 matching peptides are common to both sequences. It is not possible to tell which protein is present, or if they both are, using this method. In previous studies of the amniotic fluid protein composition,12,16,20 2D-gel electrophoresis, sometimes followed by the identification of protein spots by different immunotechniques, were the methods of choice. In a study by Liberatori et al.,16 26 AF proteins were identified by their 2D-gel pattern, among these, 8 were verified by immunoblotting and/or amino acid sequencing. Only 5 of these 26 proteins are detected with the data analysis method used in this work (with a mass cutoff of 85 kDa; see above): albumin, R-fetoprotein, R-1-antitrypsin, transferrin and R-1-microglobulin (AMBP protein). On the other hand, this analysis is unbiased since the identification is based only on the mass accuracy and retention times for the detected tryptic peptides, not by available immunochemical identification possibilities, and/or previous knowledge of common proteins in body fluids. Therefore, of the probable more than thousand proteins present in AF, the identification method used here picks the proteins that give rise to a significant tryptic peptide pattern that can be resolved and identified in the data analysis. The fact that the proteins found by Liberatori et al.16 are not identified does not mean that they are not present. If, for example, the 26 proteins found by Liberatori et al.16 are searched for within the LC-FT-ICR-MS data from Sample 1, using a mass window of 10 ppm, then tryptic peptides from the majority (18/26) of those proteins are found with a sequence covering of more than 50%. However, as the iden888

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transcription factor? part of immunoglobulin serine protease inhibitor

intracellular protein transport oxidative eosinophilic protein copper chaperon

tification algorithm used in the present work search against a larger number of proteins, many accurate peptide mass and retention time determinations are needed for statistically significant identification. If the human protein database is restricted to fewer, likely or already known proteins, then there is a higher degree of concordance between the methods. However, they would then share the same bias based on what proteins have previously been identified and for which antibodies are available. For discovery of previously unknown proteins in AF, a global, unbiased approach is preferable. For exploring protein compositions of various body fluids, LC/FT-ICR-MS has been proven to be useful as a sensitive and complementary method to 2D-gel electrophoresis and related techniques.4,7-9 New potential markers of disorder could thus be discovered. For amniotic fluid samples in particular, once shown to be compatible with LC/ESI-MS, the expansion of the sample pool, and MS/MS identification of peptides will prove if this expectation comes true.

Conclusion In this explorative study, LC/ESI-FT-ICR-MS was found to be a straightforward technique to analyze the protein composition of an amniotic fluid sample. Forty-three AF proteins were putatively identified by a bottom-up proteomic approach.

Acknowledgment. Financial support from Knut and Alice Wallenberg, the Swedish Foundation for Strategic Research, the Swedish Society for Medical Research and the Swedish Research Council (VR Grant 13123, 621-2002-5261 (J.B.) and K5104-706/2001 is gratefully acknowledged. Jonas Bergquist has a senior research position at the Swedish Research Council (VR 629-2002-6821). Supporting Information Available: The multimodal protein identification algorithm in pseudocode is available free of charge at http://pubs.acs.org. References (1) Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F., Fourier transform mass spectrometry of large molecules by electrospray ionization. Proc. Natl. Acad. Sci. U.S.A. 1989, 86(23), 9075-9078.

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Protein Composition of Amniotic Fluid (2) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray ionization for mass spectrometry of large biomolecules. Science 1989, 246, (4926), 64-71. (3) Palmblad, M.; Wetterhall, M.; Markides, K.; Håkansson, P.; Bergquist, J., Analysis of enzymatically digested proteins and protein mixtures using a 9.4 T Fourier transform ion cyclotron mass spectrometer. Rapid Commun. Mass Spectrom. 2000, 14(12), 1029-1034. (4) Palmblad, M.; Ramstro¨m, M.; Markides, K. E.; Håkansson, P.; Bergquist, J., Prediction of Chromatographic Retention and Protein Identification in Liquid Chromatography/Mass Spectrometry. Anal. Chem. 2002, 74(22), 5826-5830. (5) McLafferty, F. W.; Fridriksson, E. K.; Horn, D. M.; Lewis, M. A.; Zubarev, R. A., Biochemistry. Biomolecule mass spectrometry. Science 1999, 284(5418), 1289-1290. (6) Bergquist, J.; Palmblad, M.; Wetterhall, M.; Håkansson, P.; Markides, K. E., Peptide mapping of proteins in human body fluids using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mass Spectrom. Rev. 2002, 21(1), 2-15. (7) Bergquist, J., FTICR mass spectrometry in proteomics. Curr. Opin. Mol. Ther. 2003, 5(3), 310-314. (8) Ramstro¨m, M.; Palmblad, M.; Markides, K. E.; Håkansson, P.; Bergquist, J., Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry. Proteomics 2003, 3(2), 184-190. (9) Wu, S.-L.; Choudhary, G.; Ramstro¨m, M.; Bergquist, J.; Hancock, W. S., Evaluation of shotgun sequencing for proteomic analysis of human plasma using HPLC coupled with either ion trap or Fourier transform mass spectrometry. J. Proteome Res. 2003, 2(4), 383-393. (10) Gilbert, W. M.; Brace, R. A., Amniotic fluid volume and normal flows to and from the amniotic cavity. Semin. Perinatol. 1993, 17(3), 150-7. (11) Gulbis, B.; Gervy, C.; Jauniaux, E., Amniotic fluid biochemistry in second-trimester trisomic pregnancies: relationships to fetal organ maturation and dysfunction. Early Hum. Dev. 1998, 52(3), 211-219. (12) Bonsnes, R. W., Composition of amniotic fluid. Clin. Obstet. Gynecol. 1966, 9, (2), 440-448.

(13) Mitchell, J.; Ramstro¨m, M.; Derrick, P.; Bergquist, J. In A Comparison of Methods for Human Serum Albumin Depletion in Plasma; Analysdagarna: Go¨teborg, Sweden, 2003; Poster 65. (14) Palmblad, M.; Håkansson, K.; Håkansson, P.; Feng, X.; Cooper, H. J.; Giannakopulos, A. E.; Green, P. S.; Derrick, P. J., A 9.4 T Fourier transform ion cyclotron resonance mass spectrometer: description and performance. Eur. J. Mass Spectrom. 2000, 6(3), 267-275. (15) Nilsson, S.; Wetterhall, M.; Bergquist, J.; Nyholm, L.; Markides, K. E., A simple and robust conductive graphite coating for sheathless electrospray emitters used in capillary electrophoresis/ mass spectrometry. Rapid Commun. Mass Spectrom. 2001, 15(21), 1997-2000. (16) Liberatori, S.; Bini, L.; De Felice, C.; Magi, B.; Marzocchi, B.; Raggiaschi, R.; Frutiger, S.; Sanchez, J. C.; Wilkins, M. R.; Hughes, G.; Hochstrasser, D. F.; Bracci, R.; Pallini, V., A two-dimensional protein map of human amniotic fluid at 17 weeks’ gestation. Electrophoresis 1997, 18(15), 2816-2822. (17) Palmblad, M.; Bergquist, J., Identification and characterization of peptides and proteins using Fourier transform ion cyclotron resonance mass spectrometry. In Emerging Technologies in Protein and Genomic Material Analysis; Marko-Varga, G., Oroszlan, P., Eds.; Elsevier: 2003; Vol. 68, pp 199-240. (18) Palmblad, M. Identification and Characterization of Peptides and Proteins using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Doctoral Thesis, Uppsala University, Uppsala, available at http://urn.kb.se/resolve?urn)urn:nbn:se:uu:diva1999, 2002. (19) Ouyang, P.; Sugrue, S. P., Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex. J. Cell Biol. 1996, 135(4), 1027-1042. (20) Kronquist, K. E.; Crandall, B. F.; Cosico, L. G., Detection of novel fetal polypeptides in human amniotic fluid using twodimensional gel electrophoresis. Tumour Biol. 1984, 5(1), 15-31. (21) Boismenu, R.; Semeniuk, D.; Murgita, R. A., Purification and characterization of human and mouse recombinant alphafetoproteins expressed in Escherichia coli. Protein Expr. Purif. 1997, 10(1), 10-26.

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