Supporting Information: Protein Corona Analysis of Silver Nanoparticles Exposed to Fish Plasma Jiejun Gao 1, Lu Lin 2, Alexander Wei 2,*, and Maria S. Sepúlveda 1,* 1 Department of Forestry and Natural Resources and Bindley Biosciences Center, 2 Department of Chemistry, Purdue University, West Lafayette, Indiana, USA. *Maria S. Sepúlveda (mssepulv@purdue.edu; +1 765-496-3428), Alexander Wei (alexwei@purdue.edu; +1 765-494-5257) LC-MS/MS Analysis Methods Peptides were loaded into a trap column (20 µm x 350 mm) and washed with 2% aqueous acetonitrile (ACN), then 0.01 % formic acid (FA) in 80% ACN using a flow rate of 5 µl/min. The trap column was then switched in-line with the analytical column after 5 min. Peptides were separated using a reverse-phase Acclaim Pepmap RSLC C18 column (75 µm x 15 cm) for 120 min at a flow rate of 300 nl/min. Mobile phase A consisted of 0.01% FA in water, and mobile phase B consisted of 0.01% FA in 80% ACN. The HPLC method was comprised of a linear gradient starting from 5% B to 30% B over 80 min, then to 45% B over 11 min (t = 91 min), then 100% B over 2 min (t = 93 min). The column was held at 100% B for 5 min before being brought back to 5% B and held for 20 min. Samples were injected into the QE HF using a Nanospray Flex TM Ion source fitted with an emission tip. Data acquisition was performed by monitoring the top 20 precursors at 120,000 resolutions with an injection time of 100 milliseconds. S 1
kda Marker 250 150 100 75 50 Female VTG Males 37 VEGF 25 20 15 10 Figure S1. Western blot of plasma from adult female and male smallmouth bass sampled in the peak of spawning season from the St. Joseph s River in northern Indiana (mid- to late March). Vitellogenin (VTG) was only found in female plasma whereas vascular endothelial growth factor (VEGF) was constitutively expressed in all fish, and thus used as a reference protein. S 2
Absorbance 0.10 0.08 0.06 0.04 0.02 1 h AgNP F 1 h AgNP M 24 h AgNP F 24 h AgNP M AgNPs 0.00 400 600 800 1000 Wavelength (nm) Figure S2. UV-Vis analysis for PVP-AgNPs and of PVP-AgNPs after incubation with plasma from adult female or male smallmouth bass for 1 h or 24 h. In all cases, peak absorptions shifted to longer wavelengths after incubation with fish plasma compared with untreated PVP-AgNPs. S 3
A Particles/mL NTA 1 h 4.0 10 7 AgNPs F AgNPs AgNPs M 3.0 10 7 2.0 10 7 1.0 10 7 Plasma F Plasma M 0 0 100 200 300 400 500 Particle size (nm) B Particles/mL 4.0 10 7 NTA 24 h 3.0 10 7 2.0 10 7 1.0 10 7 AgNPs AgNPs F AgNPs M Plasma F Plasma M 0 0 100 200 300 400 500 Particle size (nm) Figure S3. Nanoparticle tracking analysis (NTA) for PVP-AgNPs after incubation with smallmouth bass plasma from adult female (F) or male (M) for 1 h (A) or 24 h (B). Shown also are NTA analyses of male and female fish plasma (no PVP-AgNPs) and untreated PVP-AgNPs. S 4
100% <20kDa 20-25kDa 25-50kDa 50-70kDa 70-100kDa 100-125kDa >125KDa 80% Protein Fraction 60% 40% 20% 0% Plasma-F PC-F, 1h PC-F, 24h Plasma-M PC-M, 1h PC-M, 24h Figure S4. Percent distribution of proteins in PCs according to size, derived from PVPcoated AgNPs exposed to adult female (F) or male (M) fish plasma for 1 h or 24 h. Protein distributions from bulk female or male plasma are shown for comparison. S 5
Figure S5. Venn diagram of proteins identified in protein corona (PC) of PVP-AgNPs incubated with fish plasma from either female (F) or male (M) smallmouth bass for 1 h or 24 h. S 6
PC-F,24h PC-F,1h PC-M,24h PC-M,1h Plasma-M Plasma-F Figure S6. Heat map of identified protein fractions (%) from protein corona (PC) isolated from PVP-AgNPs incubated with plasma from adult female (F) or male (M) smallmouth bass for 1 h or 24 h. Protein fractions are compared to plasma without PVP-AgNPs (first two columns). S 7
Figure S7. SDS-PAGE gel showing elution of proteins from protein coronas (PCs) formed after incubation of PVP-AgNPs with plasma from adult female (F) or male (M) smallmouth bass, for 1 h or 24 h, using a longer staining time. Significantly higher levels of smaller proteins (< 25 kda) were found in PCs isolated from male plasma (lanes 6 and 7), relative to those isolated from female plasma (lanes 3 and 4). S 8
Table S1. Proteins isolated from the protein corona (PC) of PVP-AgNPs after incubation with plasma from either adult female (F) or male (M) smallmouth bass, for 1 h or 24 h. Fish were collected in the peak of spawning season for northern Indiana (March). Nonredundant proteins identified through the Mascot MS/MS Ion Server (Matrix Science) are listed with their exponentially modified protein abundance index (empai) values. CaMKII: Calcium/calmodulin-dependent protein kinase; Ig: Immunoglobulin; VEGF: Vascular endothelial growth factor; VTG: Vitellogenin; ZP: Zona pellucida. Protein a PC-F-1 PC-F-24 PC-M-1 PC-M-24 Apolipoproteins A-I 0.501 0.414 0.460 0.344 A-I precursor 0.224 0.185 0.126 0.154 A-IV 0.461 0.267 0.326 B 0.224 0.185 0.059 0.154 B-100 0.316 0.076 0.141 0.063 C-II 0.699 D 0.226 E 1.951 2.047 1.098 1.340 H (beta-2-glycoprotein 1) 0.089 0.226 Proteins with Red Blood Cell Functions Coagulation Proteins alpha-1-antitrypsin 0.120 0.171 0.425 alpha-1-antitrypsin homolog 0.199 beta-fibrinogen 2.375 fibrinogen alpha chain 0.052 fibrinogen beta chain 0.527 0.566 4.148 1.466 fibrinogen beta chain precursor 0.382 0.435 0.959 fibrinogen gamma chain 0.593 0.631 0.750 0.914 fibrinogen gamma protein 0.229 0.245 0.299 gamma fibrinogen 0.555 0.950 0.715 kininogen b 0.446 S 9
kininogen-1 isoform X1 0.158 kallikrein 0.131 0.089 0.109 plasminogen 1.028 0.599 1.024 0.706 plasminogen precursor 0.120 0.208 0.154 serum amyloid A 1.806 1.491 0.297 0.869 serum amyloid A protein 0.501 0.414 0.193 0.235 vitamin K-dependent protein C 0.132 0.109 0.304 0.181 Hemoglobin beta chain 1.806 0.706 beta embryonic-2 0.564 beta-2 subunit 0.860 1.039 embryonic subunit alpha 4.521 1.699 subunit alpha-1 4.521 2.493 1.699 2.073 subunit alpha-2 c 1.885 0.882 0.601 1.294 subunit alpha-a 1.885 1.557 1.061 1.294 subunit beta d 1.143 2.634 0.308 subunit beta-1 1.002 0.718 0.564 0.299 Macroglobulin alpha-2-macroglobulin 1.911 0.686 0.344 alpha-2-macroglobulin-1 0.136 alpha-2-macroglobulin-p 0.092 0.136 Transferrins serotransferrin 0.092 0.111 0.063 transferrin 0.830 0.523 0.757 0.724 Proteins with White Blood Cell Functions Complement Proteins C1qB 0.148 C1qC 0.181 C1r/s-A isotype 0.370 0.252 0.733 C1s subcomponent 0.054 C3 2.373 1.905 1.173 2.109 C3-2 0.244 S 10
C3-2 precursor 0.087 0.104 C3 isoform X1 0.087 0.096 0.072 C4 0.026 0.022 0.052 0.018 C4-B 0.015 C5 0.290 0.272 0.772 0.253 C6 0.053 0.044 0.126 0.109 C7 0.211 0.283 0.208 0.253 C8 alpha 0.184 0.152 0.052 0.063 C8 beta 2.333 1.905 2.041 1.539 C8 gamma chain isoform X1 0.250 0.312 0.380 C9 0.606 0.327 0.957 0.498 factor B 0.109 factor H 0.052 0.063 factor I 0.063 pro-c3-1 0.343 0.316 0.193 0.326 Immunoglobulins delta heavy chain 0.119 0.098 0.104 domain-containing receptor 1 0.087 0.059 0.072 gamma heavy chain variable region 0.393 heavy chain 0.414 1.521 0.697 heavy chain BrE-3 - mouse (fragment) 0.297 heavy chain precursor 0.119 heavy chain variable region 2.070 4.768 6.063 9.060 kappa chain V region Mem5 1.818 1.285 kappa chain V-II region RPMI 6410 precursor protein 0.304 0.371 kappa chain V-IV region JI precursor 0.312 kappa light chain 0.186 lambda light chain VLJ region 0.134 lambda polypeptide 1 precursor 0.319 light chain 14.804 9.514 8.942 6.372 light chain isotype 1 1.069 0.733 S 11
light chain isotype L1 1.061 0.733 light chain precursor 0.380 light chain precursor L 1.727 1.426 1.291 0.869 light chain type 1 1.187 0.809 0.987 M heavy chain 4.271 3.527 4.861 6.997 mu heavy chain 3.770 2.896 4.601 2.009 mu heavy chain secretory form 0.448 mu heavy chain variable region 0.416 mu/tau heavy chain 2.036 1.388 2.697 tau heavy chain 0.303 0.250 0.171 0.208 Female-specific Egg Proteins VTG 1.714 1.295 VTG A 0.158 VTG Aa 0.185 0.120 VTG Ab 0.145 0.120 VTG C 0.132 0.076 VTG-1 0.119 0.087 ZP glycoprotein 2.3 precursor 0.224 0.294 Other Significant Proteins angiotensinogen 0.444 angiotensinogen precursor 0.109 CaMKII subunit alpha isoform 1 0.098 0.067 0.081 CaMKII subunit delta 0.264 0.218 0.230 0.181 ceruloplasmin 1.055 0.871 0.416 0.697 ceruloplasmin isoform X1 0.620 0.566 0.319 0.217 VEGF chain a 0.290 0.239 0.163 0.199 a Results obtained with false discovery rate (FDR) set at <5 %; b RecName: bradykinin; c AltName: alpha-2-globin or hemoglobin alpha-2 chain; d AltName: hemoglobin beta chain. S 12
Table S2. Major proteins identified from the different protein coronas (PC) with their accession number from Mascot Daemon (Matrix Science), and the number of significant peptide sequences (sig. seq.) identified for each protein. (A) PC-F, 1 h; (B) PC-F, 24 h; (C) PC-M, 1 h; (D) PC-M, 24 h. Number of sig. seq. Protein a Accession Number A B C D Apolipoproteins A-I gi 736298162 1 1 1 1 A-I precursor gi 118344628 1 1 1 1 A-IV gi 551495886 gi 734628151 3 0 2 2 gi 498985280 gi 736294062 B gi 740529756 gi 226731853 3 3 1 3 B-100 gi 657567867 gi 734622166 gi 499036286 gi 734622170 gi 736191546 gi 617302261 gi 542201992 gi 657567865 gi 432945483 21 7 11 9 C-II gi 193795860 1 0 0 0 D gi 657534183 0 0 0 1 E gi 193795858 5 5 5 5 beta-2-glycoprotein 1 (apolipoprotein H) gi 617447201 gi 323650112 0 0 1 2 Proteins with Red Blood Cell Functions Coagulation alpha-1-antitrypsin gi 397776428 gi 209981964 0 1 1 2 alpha-1-antitrypsin gi 657564670 gi 498942077 0 0 0 2 homolog antithrombin-iii gi 734648335 0 0 1 0 beta-fibrinogen gi 683842253 0 0 3 0 fibrinogen alpha chain gi 617449639 0 0 1 0 fibrinogen beta chain gi 551518621 gi 723586559 3 3 14 9 gi 373906010 gi 499043505 fibrinogen beta chain precursor gi 146447341 gi 147900556 3 3 0 8 fibrinogen gamma chain gi 617493935 gi 542187118 gi 432847401 gi 530453430 4 7 9 9 S 13
gamma fibrinogen gi 336111758 gi 46811245 0 1 2 2 kininogen b gi 46576222 0 1 0 0 kininogen-1 isoform X1 gi 499022758 1 0 0 0 kallikrein gi 551520679 0 1 1 1 plasminogen gi 551509957 gi 311223464 gi 742250469 gi 395537460 gi 410916093 gi 542250797 gi 657550476 gi 617464853 gi 507642775 13 12 26 16 plasminogen precursor gi 157278425 gi 41393105 0 2 5 3 serum amyloid A gi 401721571 gi 401721573 4 4 2 3 gi 530653512 gi 465990098 vitamin K-dependent protein C gi 657580165 gi 657811911 gi 734645142 1 1 3 2 Hemoglobin beta chain gi 157284022 3 0 0 2 beta embryonic-2 gi 47086345 0 0 2 0 beta-2 subunit gi 116488092 0 2 3 0 embryonic subunit gi 523704559 4 0 4 0 alpha subunit alpha-1 gi 432868060 4 4 4 3 subunit alpha-2 c gi 269969355 2 2 2 2 subunit alpha-a gi 734603612 2 2 2 2 subunit beta d gi 115502222 gi 122603 gi 734603610 gi 524948366 gi 225716372 0 3 9 0 subunit beta-1 gi 551527417 gi 225706810 20 21 20 1 alpha 2 Macroglobulin gi 734650793 gi 697870411 gi 734650085 gi 742195782 gi 736193097 gi 42415863 7 3 1 8 alpha 2-1 gi 323650130 0 0 0 1 alpha-2-p gi 734650797 1 0 0 2 Transferrins serotransferrin gi 6136039 1 0 2 1 S 14
transferrin gi 374431112 gi 10567299 gi 7339632 4 3 7 6 Proteins with White Blood Cell Functions Complement proteins C1qB gi 429509793 0 0 1 0 C1qC gi 429509799 0 0 0 1 C1q protein 4 gi 397511022 1 1 0 1 C1r/s-A isotype gi 67772028 0 1 1 1 C1s subcomponent gi 734635084 0 0 0 1 C3 gi 498928208 gi 657760797 gi 498929264 gi 736214174 gi 734648978 gi 657587421 gi 551507245 gi 499047883 gi 542233247 gi 657592798 gi 617471088 gi 410917688 gi 617491246 gi 657592798 gi 303305915 gi 315570434 29 32 24 41 C3 isoform X1 gi 348525110 gi 734619056 0 3 5 3 C3-2 gi 339269297 7 C3-2 precursor gi 157311657 0 3 4 0 C4 gi 684939571 gi 636571980 1 1 3 1 C4-B gi 498938159 0 0 1 0 C5 gi 499042231 gi 657589422 7 7 13 8 gi 734649196 gi 736187868 C6 gi 684939384 gi 551522012 1 1 4 3 gi 348505142 gi 465986824 C7 gi 429508173 gi 410922874 3 5 4 4 gi 6682831 gi 542188839 gi 657814026 C8 alpha gi 429508163 gi 734635553 2 2 1 1 C8 beta gi 429508165 gi 20138051 gi 328677213 gi 410924816 gi 734635551 13 15 19 14 C8 gamma chain isoform X1 gi 617385810 1 0 1 1 S 15
C9 gi 352962746 gi 408689299 gi 410903672 gi 736216070 gi 323650010 8 7 10 9 pro-c3-1 gi 573026038 9 9 9 12 factor B gi 657542545 gi 736297094 0 0 0 2 factor H gi 736308413 0 0 1 1 factor I gi 542244580 0 0 0 1 Immunoglobulins delta heavy chain gi 226860390 gi 559775728 2 2 3 0 domain-containing receptor 1 gamma heavy chain variable region heavy chain gi 677385174 0 1 1 1 gi 700652874 0 0 1 0 gi 11890707 gi 11890645 gi 2852433 0 1 3 1 heavy chain BrE-3 - gi 2137439 0 0 1 0 mouse (fragment) heavy chain precursor gi 5006471 0 0 2 0 heavy chain variable region kappa chain V region Mem5 kappa chain V-II region RPMI 6410 precursor protein gi 326632403 gi 28394637 gi 326632423 gi 28394669 gi 732549929 gi 81302953 gi 732549192 gi 732550005 3 8 13 13 gi 229367590 0 0 7 5 gi 528762398 0 0 2 2 kappa chain V-IV gi 209738284 0 0 2 0 region JI precursor kappa light chain gi 225625776 0 0 1 0 lambda light chain VLJ gi 21669497 0 0 1 0 region lambda polypeptide 1 gi 229366314 0 0 2 0 precursor light chain gi 33340577 gi 33317504 gi 34577135 gi 291165397 gi 158602744 gi 11323075 23 26 32 22 S 16
gi 33340577 gi 197700160 gi 349299 light chain isotype 1 gi 119067935 0 0 3 2 light chain isotype L1 gi 111278861 0 0 3 2 light chain precursor gi 14289265 0 0 0 2 light chain precursor L gi 37779042 5 5 6 4 light chain type 1 gi 30692167 0 2 2 2 M heavy chain gi 532579074 15 15 22 23 mu heavy chain gi 149394324 gi 322423472 gi 111228036 gi 566036242 gi 566036234 gi 334362362 11 9 18 10 mu heavy chain gi 629632982 3 0 0 0 secretory form mu heavy chain gi 372467865 0 0 3 0 variable region mu/tau heavy chain gi 566036203 gi 566036207 0 3 3 5 tau heavy chain gi 566036209 1 1 1 1 VTG Female-specific Egg Proteins gi 5852935 gi 260159577 gi 703999526 gi 687670861 gi 391353202 12 10 0 0 VTG A gi 291465276 2 0 0 0 VTG Aa gi 374923101 4 4 0 0 VTG Ab gi 326375569 4 4 0 0 VTG C gi 71011912 gi 374923105 3 2 0 0 VTG-1 gi 410921648 gi 657576314 gi 736193115 gi 657576314 4 3 0 0 ZP glycoprotein 2.3 precursor gi 185132234 2 2 0 0 Other Significant Proteins angiotensinogen gi 343459201 0 0 0 2 angiotensinogen precursor gi 213511078 0 1 0 0 S 17
CaMKIIa subunit alpha gi 395504846 0 1 1 1 isoform 1 CaMKIIa subunit delta gi 657737225 2 2 3 2 ceruloplasmin gi 657597079 gi 736207854 15 15 11 18 gi 348543049 gi 734642589 Ceruloplasmin isoform gi 551507474 gi 657739145 8 10 9 6 X1 VEGF chain A gi 157836259 1 1 1 1 a Results obtained with false discovery rate (FDR) set at <5 %; b RecName: bradykinin; c AltName: alpha-2-globin or hemoglobin alpha-2 chain; d AltName: hemoglobin beta chain. S 18