Chapter 3 Proteomic Analysis of Complex Matrices used in Human Embryonic
3.2 Results
3.4.8 Column Preparation
(PolyMicro Technologies, Phoenix, AZ) using a conventional pressure bomb (Next
Advance, Averill Park, NY). Frits were prepared by mixing 75 µL of potassium silicate
capillary end from the resulting mixture. Capillary frits were dried and hardened in a
100°C oven for 2 hours. Capillaries were then rinsed with forward and reverse flow of
methanol using the pressure bomb at 200 psi. Prior to packing, capillaries were rinsed
for 30 minutes with 0.5% formic acid for SCX and 0.5% NaOH for SAX columns.
PolySULFOETHYL A SCX beads (5 µm, 300Å, CAT# PLBMSE0503) were purchased
from PolyLC (Columbia, MD) and reconstituted in 10 mM citric acid. SAX beads were
purchased from Varian Inc. (10 µm, 1000Å, CAT# PL1451-2102, Palo Alto, CA) and
reconstituted in 10 mM NH4OH. SCX and SAX columns were packed to a length of ~3
cm for all experiments. All packing was done at a pressure of 200 psi until the desired
column length was obtained, then pressurized to 1000 psi to complete packing. Packed
columns were rinsed with solvents for conditioning as recommended by the
manufacturer.
3.4.9 Peptide Fractionation – Peptides were fractionated with SCX and SAX columns
using a CapLC system (Waters, Milford, MA) with a flow rate of 2 µL/min. Mobile phase
A was composed of 10mM citric acid at a pH of ~2.3, mobile phase B was pH 11.4 10
mM NH4OH, and mobile phase C was 500 mM ammonium acetate. Mobile phases
were all prepared fresh prior to fractionation to ensure the stability of pH from run to run.
Fractionation with pH was carried out using a 120 minute gradient from pH 2 to 12 for
SCX, and 12 to 2 for SAX. Salt fractionation was carried using the following steps:
0 mM, 7.5 mM, 15 mM, 30 mM, 45 mM, 60 mM, 75 mM, 90 mM, 105 mM, 120 mM, 150
mM, 300 mM, 500 mM ammonium acetate over 120 minutes. Each fraction was
collected offline in a volume of 20 µL. Fractions were dried using a SpeedVac, and
3.4.10 MS Data Acquisition – Fractions were injected and separated using a
nanoAcquity system (Waters, Milford, MA) equipped with a 25 cm x 75 µm I.D. C18
column. Fractions were separated using a 1 to 40% ACN gradient over 90 minutes at a
flow rate of 300 nL/min. MS analysis was done on a Q-ToF Ultima (Micromass/Waters)
using data-dependent acquisition with selection of the four top precursor masses per
survey scan. Survey scans were set at 1s, and MS/MS acquisition set at 1s or 6000
cps TIC cut-off. Exclusion lists were generated using in-house software that
automatically adds 0.7amu to the precursor masses selected in previous runs and
outputs a file also containing retention time information. Each fraction was analyzed for
a single exclusion round (2 injections total), adjusting the injection volume dependent on
signal intensity in the previous run. In analyses of Matrigel™, each fraction was injected
a total of 7 times (6 exclusion rounds).
3.4.11 MS Data analysis – Data analysis for all matrices tested was performed in
PEAKS 5.3 software (Bioinformatic Solutions Inc., Waterloo, Canada) [79, 80]. After
import into PEAKS, MS/MS spectra from raw data files were refined using the following
settings: merge spectra – true (100 ppm mass tolerance, 60 second retention time
tolerance), correct precursor mass – true, determine precursor charge state – true
(minimum charge +2, maximum charge +5), spectral quality filter – true (0.65 threshold),
centroid, deisotope, and deconvolute – true. Resulting MS/MS spectra were then de
novo sequenced using the following parameters: parent monoisotopic mass tolerance –
100ppm, fragment monoisotopic mass tolerance – 0.15 daltons, enzyme specificity –
semi-Trypsin, fixed modifications – carbamidomethylation, variable modifications – N-
searched against the UniProt sequence database (Human taxonomy specified, 20236
total entries or Mouse taxonomy specified, 16376 total entries) using the following
parameters: parent monoisotopic mass tolerance – 100 ppm, fragment monoisotopic
mass tolerance – 0.15 daltons, enzyme specificity – semi-Trypsin, fixed modifications –
carbamidomethylation, variable modifications – N-terminal acetylation and oxidized
methionine, estimate false-discovery rate – true.
3.4.12 Protein identification – Resultant protein identification data from the database
search were processed using PEAKS 5.3 software [81]. Identifications were filtered by
assigned score to give a <1.0% false-discovery rate for peptide-spectral matches based
on estimation from a decoy-fusion method of database search. Identified proteins were
also required to be matched by >2 unique peptides. Proteins that contained similar
peptides and could not be differentiated based on MS/MS analysis alone were grouped
to satisfy the principles of parsimony. For all matrices analyzed, keratin hits were
manually removed from the final datasets.
3.4.13 Real-time PCR – RNA was purified using Trizol reagent according to the
manufacturer’s instructions (Invitrogen) or with the 5 Prime PerfectPure RNA Extraction
Kit (5 Prime, Gaithersburg, MD). After NanoDrop quantification, 1 µg of cDNA was
synthesized using the High Capacity cDNA Reverse Transcription Kit with RNase
inhibitor (Invitrogen). Real-time PCR was performed using the TaqMan® Universal
PCR Master Mix (Invitrogen). Samples were incubated at 50°C for 2 minutes followed
by 10 minutes at 95°C. Samples were then amplified at 95°C for 15 seconds followed
protein RPLPO as a positive control. All primers were obtained from Invitrogen and
assay information can be found in Appendix Table 1.15. Each biological replicate was
run in triplicate for every marker assayed. All reagents were used according to the
manufacturer’s instructions.
3.4.14 Immunofluorescence – Passage 10 H9 and 5 CA1 hESCs grown on fibronectin
were plated in wells of a 24-well dish coated with the same matrix. After 4 days of
culture in mTeSR1 medium cells were rinsed and fixed with 4% para-formaldehyde
containing 20 mM sucrose. Prior to staining, cells were blocked using the Dako Serum
Free Protein Block solution (Dako, Cambridgeshire, UK). Oct-4 (clone 10H11.2),
SSEA-4 (clone MC-813-70), and SSEA-1 (clone MC-480) primary antibodies were
obtained from Millipore (Billerica, MA). Cells for Oct-4 labeling were permeabilized
using a 0.1% Triton X-100 solution prior to blocking. Cells were labeled with primary
antibodies for Oct-4 or SSEA-4 as undifferentiated markers and SSEA-1 for
differentiated cells all at concentrations of 2 μg/mL for 60 minutes at room temperature.
Secondary Alexa-fluor 488 goat anti-mouse IgG and Alexa-fluor 568 goat anti-mouse
IgM were obtained from Invitrogen (Carlsbad, CA). Secondary antibodies were probed at concentrations of 1 μg/mL for 60 minutes at room temperature. All products were used according to manufacturer’s instructions.
3.4 References
[1] Lin, C. Q., Bissell, M. J., Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J
1993, 7, 737-743.
[2] Martin, G. R., Kleinman, H. K., Extracellular matrix proteins give new life to cell culture. Hepatology
1981, 1, 264-266.
[3] Kleinman, H. K., Graf, J., Iwamoto, Y., Kitten, G. T., Ogle, R. C., Sasaki, M., et al., Role of basement membranes in cell differentiation. Ann N Y Acad Sci 1987, 513, 134-145.
[4] Kleinman, H. K., Klebe, R. J., Martin, G. R., Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol 1981, 88, 473-485.
[5] Erickson, A. C., Couchman, J. R., Still more complexity in mammalian basement membranes. J Histochem Cytochem 2000, 48, 1291-1306.
[6] Sawada, N., Tomomura, A., Sattler, C. A., Sattler, G. L., Kleinman, H. K., Pitot, H. C., Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture. Exp Cell Res 1986,
167, 458-470.
[7] Kleinman, H. K., Cannon, F. B., Laurie, G. W., Hassell, J. R., Aumailley, M., Terranova, V. P., et al., Biological activities of laminin. J Cell Biochem 1985, 27, 317-325.
[8] Kleinman, H. K., Martin, G. R., Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol 2005, 15, 378-386.
[9] Kleinman, H. K., McGarvey, M. L., Liotta, L. A., Robey, P. G., Tryggvason, K., Martin, G. R., Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 1982, 21, 6188-6193.
[10] Orkin, R. W., Gehron, P., McGoodwin, E. B., Martin, G. R., Valentine, T., Swarm, R., A murine tumor producing a matrix of basement membrane. J Exp Med 1977, 145, 204-220.
[11] Vukicevic, S., Kleinman, H. K., Luyten, F. P., Roberts, A. B., Roche, N. S., Reddi, A. H., Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res 1992, 202, 1-8.
[12] Lei, T., Jacob, S., Ajil-Zaraa, I., Dubuisson, J. B., Irion, O., Jaconi, M., et al., Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges. Cell Research 2007, 17, 682-688.
[13] Skottman, H., Narkilahti, S., Hovatta, O., Challenges and approaches to the culture of pluripotent human embryonic stem cells. Regen Med 2007, 2, 265-273.
[14] Gerecht, S., Burdick, J. A., Ferreira, L. S., Townsend, S. A., Langer, R., Vunjak-Novakovic, G., Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells.
Proc Natl Acad Sci U S A 2007, 104, 11298-11303.
[15] Braam, S. R., Zeinstra, L., Litjens, S., Ward-van Oostwaard, D., van den Brink, S., van Laake, L., et al., Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alpha V beta 5 integrin. Stem Cells 2008, 26, 2257-2265.
[16] Hakala, H., Rajala, K., Ojala, M., Panula, S., Areva, S., Kellomaki, M., et al., Comparison of
Biomaterials and Extracellular Matrices as a Culture Platform for Multiple, Independently Derived Human Embryonic Stem Cell Lines. Tissue Engineering Part A 2009, 15, 1775-1785.
[17] Ludwig, T. E., Levenstein, M. E., Jones, J. M., Berggren, W. T., Mitchen, E. R., Frane, J. L., et al., Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 2006, 24, 185-187. [18] Amit, M., Shariki, C., Margulets, V., Itskovitz-Eldor, J., Feeder layer- and serum-free culture of human embryonic stem cells. Biology of Reproduction 2004, 70, 837-845.
[19] Manton, K. J., Richards, S., Van Lonkhuyzen, D., Cormack, L., Leavesley, D., Upton, Z., A chimeric vitronectin: igf-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells.
Stem Cells Dev 2010, 19, 1297-1305.
[20] Mei, Y., Saha, K., Bogatyrev, S. R., Yang, J., Hook, A. L., Kalcioglu, Z. I., et al., Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat Mater 2010, 9, 768- 778.
[21] Klim, J. R., Li, L. Y., Wrighton, P. J., Piekarczyk, M. S., Kiessling, L. L., A defined glycosaminoglycan- binding substratum for human pluripotent stem cells. Nature Methods 2010, 7, 989-U972.
[22] Harb, N., Archer, T. K., Sato, N., The Rho-Rock-Myosin Signaling Axis Determines Cell-Cell Integrity of Self-Renewing Pluripotent Stem Cells. Plos One 2008, 3, -.
[23] Miyazaki, T., Futaki, S., Hasegawa, K., Kawasaki, M., Sanzen, N., Hayashi, M., et al., Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells.
Biochemical and Biophysical Research Communications 2008, 375, 27-32.
[24] Vuoristo, S., Virtanen, I., Takkunen, M., Palgi, J., Kikkawa, Y., Rousselle, P., et al., Laminin isoforms in human embryonic stem cells: synthesis, receptor usage and growth support. Journal of Cellular and Molecular Medicine 2009, 13, 2622-2633.
[25] Derda, R., Li, L., Orner, B. P., Lewis, R. L., Thomson, J. A., Kiessling, L. L., Defined substrates for human embryonic stem cell growth identified from surface arrays. ACS Chem Biol 2007, 2, 347-355. [26] Brafman, D. A., Chang, C. W., Fernandez, A., Willert, K., Varghese, S., Chien, S., Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces. Biomaterials 2010, 31, 9135-9144. [27] Kolhar, P., Kotamraju, V. R., Hikita, S. T., Clegg, D. O., Ruoslahti, E., Synthetic surfaces for human embryonic stem cell culture. J Biotechnol 2010, 146, 143-146.
[28] Melkoumian, Z., Weber, J. L., Weber, D. M., Fadeev, A. G., Zhou, Y., Dolley-Sonneville, P., et al., Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat Biotechnol 2010, 28, 606-610.
[29] Steiner, D., Khaner, H., Cohen, M., Even-Ram, S., Gil, Y., Itsykson, P., et al., Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 2010, 28, 361-364.
[30] Meng, G. L., Liu, S. Y., Li, X. Y., Krawetz, R., Rancourt, D. E., Extracellular Matrix Isolated From Foreskin Fibroblasts Supports Long-Term Xeno-Free Human Embryonic Stem Cell Culture. Stem Cells and Development 2010, 19, 547-556.
[31] Amit, M., Margulets, V., Segev, H., Shariki, K., Laevsky, I., Coleman, R., et al., Human feeder layers for human embryonic stem cells. Biology of Reproduction 2003, 68, 2150-2156.
[32] Chen, H. F., Chuang, C. Y., Shieh, Y. K., Chang, H. W., Ho, H. N., Kuo, H. C., Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions. Human Reproduction 2009, 24, 1114-1125.
[33] Hovatta, O., Mikkola, M., Gertow, K., Stromberg, A. M., Inzunza, J., Hreinsson, J., et al., A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Human Reproduction 2003, 18, 1404-1409.
[34] Kibschull, M., Mileikovsky, M., Nagy, A., Lye, S. J., Human Embryonic Fibroblast Lines Provide Enhanced Support of Human Embryonic Stem Cells in Xeno-Free Culture Conditions. Reproductive Sciences 2009, 16, 282a-282a.
[35] Swistowski, A., Peng, J., Han, Y., Swistowska, A. M., Rao, M. S., Zeng, X., Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them. Plos One 2009, 4, e6233.
[36] Hakala, H., Rajala, K., Ojala, M., Panula, S., Areva, S., Kellomaki, M., et al., Comparison of
Biomaterials and Extracellular Matrices as a Culture Platform for Multiple, Independently Derived Human Embryonic Stem Cell Lines. Tissue Eng Part A 2009.
[37] Lu, J., Hou, R. H., Booth, C. J., Yang, S. H., Snyder, M., Defined culture conditions of human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America
2006, 103, 5688-5693.
[38] Mallon, B. S., Park, K. Y., Chen, K. G., Hamilton, R. S., McKay, R. D. G., Toward xeno-free culture of human embryonic stem cells. International Journal of Biochemistry & Cell Biology 2006, 38, 1063-1075. [39] Li, Q., Wang, J., Shahani, S., Sun, D. D., Sharma, B., Elisseeff, J. H., et al., Biodegradable and photocrosslinkable polyphosphoester hydrogel. Biomaterials 2006, 27, 1027-1034.
[40] Chen, S. S., Fitzgerald, W., Zimmerberg, J., Kleinman, H. K., Margolis, L., Cell-cell and cell- extracellular matrix interactions regulate embryonic stem cell differentiation. Stem Cells 2007, 25, 553- 561.
[41] Philp, D., Chen, S. S., Fitzgerald, W., Orenstein, J., Margolis, L., Kleinman, H. K., Complex extracellular matrices promote tissue-specific stem cell differentiation. Stem Cells 2005, 23, 288-296. [42] McCloskey, K. E., Gilroy, M. E., Nerem, R. M., Use of embryonic stem cell-derived endothelial cells as a cell source to generate vessel structures in vitro. Tissue Engineering 2005, 11, 497-505.
[43] Kim, M. S., Hwang, N. S., Lee, J., Kim, T. K., Leong, K., Shamblott, M. J., et al., Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells 2005, 23, 113-123.
[44] Battista, S., Guarnieri, D., Borselli, C., Zeppetelli, S., Borzacchiello, A., Mayol, L., et al., The effect of matrix composition of 3D constructs on embryonic stem cell differentiation. Biomaterials 2005, 26, 6194- 6207.
[45] Baxter, M. A., Camarasa, M. V., Bates, N., Small, F., Murray, P., Edgar, D., et al., Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines. Stem Cell Res 2009.
[46] Bendall, S. C., Hughes, C., Campbell, J. L., Stewart, M. H., Pittock, P., Liu, S., et al., An Enhanced Mass Spectrometry Approach Reveals Human Embryonic Stem Cell Growth Factors in Culture. Molecular & Cellular Proteomics 2009, 8, 421-432.
[47] Prowse, A. B. J., McQuade, L. R., Bryant, K. J., Marcal, H., Gray, P. P., Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media. Journal of Proteome Research 2007, 6, 3796-3807.
[48] Prowse, A. B. J., McQuade, L. R., Bryant, K. J., Van Dyk, D. D., Tuch, B. E., Gray, P. P., A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells. Proteomics 2005, 5, 978-989.
[49] Lim, J. W. E., Bodnar, A., Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2002, 2, 1187-1203. [50] Chin, A. C. P., Fong, W. J., Goh, L. T., Philp, R., Oh, S. K. W., Choo, A. B. H., Identification of proteins from feeder conditioned medium that support human embryonic stem cells. Journal of Biotechnology 2007, 130, 320-328.
[51] Faca, V., Pitteri, S. J., Newcomb, L., Glukhova, V., Phanstiel, D., Krasnoselsky, A., et al.,
Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. J Proteome Res 2007, 6, 3558-3565.
[52] de Godoy, L. M., Olsen, J. V., de Souza, G. A., Li, G., Mortensen, P., Mann, M., Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system. Genome Biol 2006, 7, R50.
[53] Washburn, M. P., Wolters, D., Yates, J. R., 3rd, Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001, 19, 242-247.
[54] Fang, Y., Robinson, D. P., Foster, L. J., Quantitative analysis of proteome coverage and recovery rates for upstream fractionation methods in proteomics. J Proteome Res 2010, 9, 1902-1912.
[55] Wang, N., Li, L., Exploring the precursor ion exclusion feature of liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry for improving protein identification in shotgun proteome analysis. Anal Chem 2008, 80, 4696-4710.
[56] Klimanskaya, I., Chung, Y., Meisner, L., Johnson, J., West, M. D., Lanza, R., Human embryonic stem cells derived without feeder cells. Lancet 2005, 365, 1636-1641.
[57] Bendall, S. C., Stewart, M. H., Menendez, P., George, D., Vijayaragavan, K., Werbowetski-Ogilvie, T., et al., IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 2007, 448, 1015-1021.
[58] Fava, R. A., McClure, D. B., Fibronectin-associated transforming growth factor. J Cell Physiol 1987,
131, 184-189.
[59] Ruoslahti, E., Yamaguchi, Y., Hildebrand, A., Border, W. A., Extracellular matrix/growth factor interactions. Cold Spring Harb Symp Quant Biol 1992, 57, 309-315.
[60] Nakamura, T., Sugino, K., Titani, K., Sugino, H., Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells. J Biol Chem 1991, 266, 19432- 19437.
[61] Murphy-Ullrich, J. E., Schultz-Cherry, S., Hook, M., Transforming growth factor-beta complexes with thrombospondin. Mol Biol Cell 1992, 3, 181-188.
[62] Rahman, S., Patel, Y., Murray, J., Patel, K. V., Sumathipala, R., Sobel, M., et al., Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells. BMC Cell Biol 2005, 6, 8.
[63] Wijelath, E. S., Rahman, S., Namekata, M., Murray, J., Nishimura, T., Mostafavi-Pour, Z., et al., Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain - Enhancement of
VEGF biological activity by a singular growth factor/matrix protein synergism. Circulation Research 2006,
99, 853-860.
[64] Golombick, T., Dajee, D., Bezwoda, W. R., Extracellular matrix interactions. 2: Extracellular matrix structure is important for growth factor localization and function. In Vitro Cell Dev Biol Anim 1995, 31, 396-403.
[65] Taipale, J., Keski-Oja, J., Growth factors in the extracellular matrix. FASEB J 1997, 11, 51-59. [66] Dinbergs, I. D., Brown, L., Edelman, E. R., Cellular response to transforming growth factor-beta1 and basic fibroblast growth factor depends on release kinetics and extracellular matrix interactions. J Biol Chem 1996, 271, 29822-29829.
[67] Butzow, R., Fukushima, D., Twardzik, D. R., Ruoslahti, E., A 60-kD protein mediates the binding of transforming growth factor-beta to cell surface and extracellular matrix proteoglycans. J Cell Biol 1993,
122, 721-727.
[68] Evseenko, D., Schenke-Layland, K., Dravid, G., Zhu, Y. H., Hao, Q. L., Scholes, J., et al., Identification of the Critical Extracellular Matrix Proteins that Promote Human Embryonic Stem Cell Assembly. Stem Cells and Development 2009, 18, 919-927.
[69] Li, Y., Powell, S., Brunette, E., Lebkowski, J., Mandalam, R., Expansion of human embryonic stem