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CHAPTER 7.PROTEIN IDENTIFICATION IN DEPOSITS FROM BLOOD PLASMA ON PEG
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7.2 Identification of proteins in blood plasma deposits
37 different proteins were identified in plasma deposits of all the tested surfaces. The deposits on SAM, PEG-OH, PEG-COOH, PEG-COOH, poly(MeOEGMA), and poly(HOEGMA) contained 27, 24, 24, 20, 11, and 7 different proteins, respectively. The decreasing number of different proteins contained in the deposits on different types of surfaces (SAM, end-tethered PEG, and polymer brushes) was accompanied by a decrease in the total deposited mass (Figure C7-2)
There was no observable relationship between the deposit composition and molecular weight of individual proteins. The protein assemblies, which were deposited on the best anti-fouling surfaces, poly(HOEGMA) and poly(MeOEGMA) brushes, consisted only of apolipoprotein A-I (ApoA-I, Mw: 31 kDa), apolipoprotein B-100 (ApoB-100, Mw: 516 kDa), complement C3 (C3, Mw: 187 kDa), Fbg (Mw: 340 kDa), histidine-rich glycoprotein (HRGP, Mw: 60 kDa), Ig mu chain C region (Ig muC, Mw: 49 kDa), and HSA (Mw: 69 kDa). The proteins were present in the deposits on all other surfaces tested in this work suggesting some specificity for PEG. These results are in good agreement with a recent study of Walkey et al. finding the same proteins among 12 other proteins on PEG (Mw:5000 g·mol-1) tethered to gold nanoparticles.[9]
Remarkably, HSA and Fbg were found in the plasma deposits on poly(HOEGMA) and poly(MeOEGMA) brushes even if these surfaces suppressed adsorption of these proteins from single protein solutions. The adsorption of HSA and Fbg as part of the plasma deposit seems to be mediated by some other plasma proteins. A possible mechanism might consist in the adsorption of Fbg on PEG structures with a distorted conformation by previous adsorption of other proteins or by some synergic interaction of Fbg with some specific protein. Histidine-rich glycoprotein (HRGP) seems to be a conceivable partner of Fbg, as it can bind to it specifically.[10]
Interestingly the deposits from ultrafiltrated blood plasma lacked not only high molecular weight proteins (ApoB-100 and Fbg) but also some proteins of much smaller molecular weights than the filter cut-off which were present in deposits of whole blood plasma on SAM, and tethered PEG chain coatings (Table C7-2). This indicates that adsorption of these small proteins was mediated by some of the bigger ones removed by filtration.
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Table C7-1. Identified proteins from human blood plasma deposits on PEG-based surfaces using LC-MS/MS analysis.
Table C7-2. Identified proteins from ultrafiltrated human blood plasma deposits on PEG-based surfaces using LC-MS/MS analysis.
The adsorption of ApoA-I, ApoB-100, C3, HRGP, and Ig muC seems to be a distinct general feature of the plasma fouling on PEG-based surfaces. Other independent studies on
1 Annexin A1 P04083 38,714 X
2 Alpha-1-antitrypsin P01009 46,737 X X X X
3 Alpha-1-antichymotrypsin P01011 47,651 X
4 Apolipoprotein A-I P02647 30,778 X X X X X X
5 Apolipoprotein B-100 P04114 515,563 X X X X X X
6 Apolipoprotein C-III P02656 10,852 X X
7 Apolipoprotein E P02649 36,154 X X X X X
8 C4b-binding protein alpha chain P04003 67,033 X X X
9 Clusterin P10909 52,495 X X
10 Coagulation factor V P12259 251,671 X X X X
11 Complement C1q subcomponent subunit B P02746 26,459 X
12 Complement C1s subcomponent P09871 76,684 X X
13 Complement C3 P01024 187,148 X X X X X X
14 Complement C4-A P0C0L4 192,771 X X X X X
15 Complement C4-B P0C0L5 192,793 X X X X X
16 Complement C5 P01031 188,305 X
17 Complement component C9 P02748 63,173 X X X
18 Complement factor H P08603 139,096 X X X X
19 Desmoglein-1 Q02413 113,716 X
20 Desmoplakin P15924 331,774 X
21 Fibrinogen alpha chain P02671 94,973 X X X X X X
22 Fibrinogen beta chain P02675 55,928 X X X X X X
23 Fibrinogen gamma chain P02679 51,512 X X X X X X
24 Fibronectin P02751 262,607 X X
25 Fibulin-1 P23142 77,214 X
26 Histidine-rich glycoprotein P04196 59,578 X X X X X X
27 Ig alpha-1 chain C region P01876 37,655 X X X X
28 Ig gamma-1 chain C region P01857 36,106 X X X
29 Ig kappa chain C region P01834 11,609 X X X
30 Ig mu chain C region P01871 49,307 X X X X X X
31 Inter-alpha-trypsin inhibitor heavy chain H4 Q14624 103,357 X X
32 Lactotransferrin P02788 78,182 X
33 Lipopolysaccharide-binding protein P18428 53,384 X X
34 Kininogen-1 P01042 71,957 X
35 Myeloperoxidase P05164 83,869 X
36 Plasma serine protease inhibitor P05154 45,702 X
37 Proteoglycan 4 Q92954 151,077 X
38 Serum albumin P02768 69,367 X X X X X X
39 Vitronectin P04004 54,306 X X X X
MeOEGMA mPEG PEG-OH PEG-COOH HOEGMA
UniProt AC
Identified proteins Mass (Da) SAM
1 Alpha-1-antitrypsin P01009 46.737 X
2 Apolipoprotein A-I P02647 30.778 X X X X X X
3 Apolipoprotein C-III P02656 10.852 X X
4 Apolipoprotein E P02649 36.154 X X X X
5 C4b-binding protein alpha chain P04003 67.033 X
6 Clusterin P10909 52.495 X X X
7 Coagulation factor V P12259 251.671 X X X
8 Complement C3 P01024 187.148 X X X X X
9 Complement C4-A P0C0L4 192.771 X X X X X
10 Complement C4-B P0C0L5 192.793 X X X X X
11 Complement component C9 P02748 63.173 X
12 Desmoglein-1 Q02413 113.716 X
13 Histidine-rich glycoprotein P04196 59.578 X X X X
14 Ig alpha-1 chain C region P01876 37.655 X X
15 Ig gamma-1 chain C region P01857 36.106 X X X X
16 Ig kappa chain C region P01834 11.609 X X X X X
17 Ig mu chain C region P01871 49.307 X X X X X
18 Inter-alpha-trypsin inhibitor heavy chain H4 Q14624 103.357 X X
19 Serum albumin P02768 69.367 X X X X X X
20 Vitronectin P04004 54.306 X
Mass (Da) SAM mPEG PEG-OH PEG-COOH HOEGMA MeOEGMA UniProt AC
Identified proteins
CHAPTER 7.PROTEIN IDENTIFICATION IN DEPOSITS FROM BLOOD PLASMA ON PEG
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identifying proteins deposited on PEG surfaces with different architectures have also found them.[3, 9, 11, 12] The lower fouling on poly(MeOEGMA) bearing a hydrophobic methacrylate backbone and metoxy-capped lateral chains compared to the more hydrophilic polyether backbone of end-tethered PEG-OH suggest that hydrophobic interactions did not play a significant role. PEG resistance to fouling can be associated with the high enthalpy penalty caused by the dehydration suffered when a protein adsorbs on PEG. The high enthalpy change upon dehydration is due to the strong interactions between lone pairs of electrons on the oxygen atom of the ether groups and the electron-poor protons of water. Theoretically, the water can be replaced by stronger interactions between the ether oxygen, which is hydrogen bond acceptor, and hydrogen bond acceptor NH3+ groups of some amino acid residues or NH groups of the main-peptidic chain.
Interestingly, the very same principle promoting a very important reduction of fouling might be the cause of adsorption of the previously mentioned seven proteins mediated by biospecific interactions.
Recent X-rays diffraction studies evidenced that PEG interacts with protein surface[13, 14] by four mechanisms:
i) multiple coordination contacts to positively charged lysine, arginine and histidine residues
ii) hydrogen bonds to side chains of amino acid residues iii) hydrogen bond with amidic backbone of proteins
iv) coordination fixing some cation from solution and forming hydrophobic interactions between the outer envelope of the complexed cation with prevailing hydrophobic areas at the protein surface.[13, 14]
The very flexible nature and variety of possible interactions of PEG molecules result in its ability to penetrate into intra and inter molecular cleft in proteins linking or modifying regions of protein surface. These specific interactions might be responsible for the fouling of some specific protein even in best PEG-based surfaces. The adsorption of these proteins can mediate a subsequent adsorption of other components.
Most research so far has focused in explaining protein surface interaction solely by simplified models considering only the properties of the surface. This research clearly shows that a comprehensive understanding of fouling requires models considering both surface and proteins as well as biospecific interactions.
7.3 Summary
The presence of a protein in plasma deposits had no correlation with molecular weight. This confirms our previous results (Chapter 5) indicating that proteins did not penetrate the brush.
7-7
The mass of the deposits from blood plasma on the studied surfaces decreased in the following order: SAMs > PEGs > poly(OEGMA) brushes probably due to a more efficient water barrier. The reduction in the mass of the deposits was accompanied by a decrease in the number of adsorbed proteins.
No significant effect of PEG end-groups on the fouling suggest that hydrophobic interaction plays little part on the fouling process.
The adsorption of HSA and Fbg on poly(OEGMA) brushes seems to be mediated by the preliminary absorption of other proteins. A similar effect was observed on the deposits from blood plasma depleted from high molecular weight compounds, where not only big proteins were ruled out of the deposits but also smaller ones, whose adsorption was probably linked to the adsorption of the larger ones.
Seven proteins were found in the deposits on the best antifouling surfaces, poly(HOEGMA) and poly(MeOEGMA). These proteins were present in the deposits of all PEG-base coatings studied. The ubiquity of these proteins on PEG-based coatings together with previous X-Rays studies showing the interaction of PEG with protein surfaces, suggest that some biospecific interactions mediate their adsorption.
From these conclusions two corollaries can be extracted:
i) A comprehensive understanding of fouling cannot be achieved if only properties of the surfaces are considered.
ii) Due to some biospecific interaction any PEG coating will suffer fouling, ergo the design of non-fouling surfaces requires the use of new monomers.
The last corollary motivated the research presented in Chapter 8. The selection of the new monomers is based on the lessons drawn from Chapter 4 to 7. The new surfaces should fulfill the following requisites:
i) Electroneutral ii) High surface density
iii) A brush architecture (entropic barrier) iv) Highly wettable (hydration barrier)
7.4 References
[1] L. D. Unsworth, H. Sheardown, J. L. Brash, Langmuir 2005, 21, 1036.
[2] L. D. Unsworth, H. Sheardown, J. L. Brash, Biomaterials 2005, 26, 5927.
[3] M. E. Price, R. M. Cornelius, J. L. Brash, Biochimica Et Biophysica Acta-Biomembranes 2001, 1512, 191.
[4] S. I. Jeon, J. D. Andrade, J. Colloid Interface Sci. 1991, 142, 159.
CHAPTER 7.PROTEIN IDENTIFICATION IN DEPOSITS FROM BLOOD PLASMA ON PEG
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[5] C. Rodriguez-Emmenegger, E. Brynda, T. Riedel, M. Houska, V. Šubr, A. Bologna Alles, E. Hasan, J. E. Gautrot, W. T. S. Huck, Macromol. Rapid Commun. 2011, 32, 952.
[6] C. Rodriguez-Emmenegger, E. Brynda, T. Riedel, Z. Sedlakova, M. Houska, A. B.
Alles, Langmuir 2009, 25, 6328.
[7] S. Jiang, Z. Cao, Adv. Mater. 2010, 22, 920.
[8] N. Barnthip, P. Parhi, A. Golas, E. A. Vogler, Biomaterials 2009, 30, 6495.
[9] C. D. Walkey, J. B. Olsen, H. Guo, A. Emili, W. C. W. Chan, J. Am. Chem. Soc. 2011, DOI: 10.1021/ja2084338.
[10] A. L. Jones, M. D. Hulett, C. R. Parish, Immunol. Cell Biol. 2005, 83, 106.
[11] R. Gref, M. Luck, P. Quellec, M. Marchand, E. Dellacherie, S. Harnisch, T. Blunk, R.
H. Muller, Colloids and Surfaces B-Biointerfaces 2000, 18, 301.
[12] R. M. Cornelius, J. MacRi, J. L. Brash, Journal of Biomedical Materials Research - Part A 2011, 99 A, 109.
[13] J. Hašek, Zeitschrift fur Kristallographie, Supplement 2006, 2, 613.
[14] J. Hašek, Journal of Synchrotron Radiation 2011, 18, 50.
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