Supporting Information: Protein Corona Analysis of Silver Nanoparticles Exposed to Fish Plasma

Transcription

1 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; ), Alexander Wei (alexwei@purdue.edu; ) 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

2 kda Marker Female VTG Males 37 VEGF 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

3 Absorbance h AgNP F 1 h AgNP M 24 h AgNP F 24 h AgNP M AgNPs 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

4 A Particles/mL NTA 1 h AgNPs F AgNPs AgNPs M Plasma F Plasma M Particle size (nm) B Particles/mL NTA 24 h AgNPs AgNPs F AgNPs M Plasma F Plasma M 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

5 100% <20kDa 20-25kDa 25-50kDa 50-70kDa kDa kDa >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

6 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

7 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

8 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

9 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 A-I precursor A-IV B B C-II D E H (beta-2-glycoprotein 1) Proteins with Red Blood Cell Functions Coagulation Proteins alpha-1-antitrypsin alpha-1-antitrypsin homolog beta-fibrinogen fibrinogen alpha chain fibrinogen beta chain fibrinogen beta chain precursor fibrinogen gamma chain fibrinogen gamma protein gamma fibrinogen kininogen b S 9

10 kininogen-1 isoform X kallikrein plasminogen plasminogen precursor serum amyloid A serum amyloid A protein vitamin K-dependent protein C Hemoglobin beta chain beta embryonic beta-2 subunit embryonic subunit alpha subunit alpha subunit alpha-2 c subunit alpha-a subunit beta d subunit beta Macroglobulin alpha-2-macroglobulin alpha-2-macroglobulin alpha-2-macroglobulin-p Transferrins serotransferrin transferrin Proteins with White Blood Cell Functions Complement Proteins C1qB C1qC C1r/s-A isotype C1s subcomponent C C S 10

11 C3-2 precursor C3 isoform X C C4-B C C C C8 alpha C8 beta C8 gamma chain isoform X C factor B factor H factor I pro-c Immunoglobulins delta heavy chain domain-containing receptor gamma heavy chain variable region heavy chain heavy chain BrE-3 - mouse (fragment) heavy chain precursor heavy chain variable region kappa chain V region Mem kappa chain V-II region RPMI 6410 precursor protein kappa chain V-IV region JI precursor kappa light chain lambda light chain VLJ region lambda polypeptide 1 precursor light chain light chain isotype S 11

12 light chain isotype L light chain precursor light chain precursor L light chain type M heavy chain mu heavy chain mu heavy chain secretory form mu heavy chain variable region mu/tau heavy chain tau heavy chain Female-specific Egg Proteins VTG VTG A VTG Aa VTG Ab VTG C VTG ZP glycoprotein 2.3 precursor Other Significant Proteins angiotensinogen angiotensinogen precursor CaMKII subunit alpha isoform CaMKII subunit delta ceruloplasmin ceruloplasmin isoform X VEGF chain a 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

13 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 A-I precursor gi A-IV gi gi gi gi B gi gi B-100 gi gi gi gi gi gi gi gi gi C-II gi D gi E gi beta-2-glycoprotein 1 (apolipoprotein H) gi gi Proteins with Red Blood Cell Functions Coagulation alpha-1-antitrypsin gi gi alpha-1-antitrypsin gi gi homolog antithrombin-iii gi beta-fibrinogen gi fibrinogen alpha chain gi fibrinogen beta chain gi gi gi gi fibrinogen beta chain precursor gi gi fibrinogen gamma chain gi gi gi gi S 13

14 gamma fibrinogen gi gi kininogen b gi kininogen-1 isoform X1 gi kallikrein gi plasminogen gi gi gi gi gi gi gi gi gi plasminogen precursor gi gi serum amyloid A gi gi gi gi vitamin K-dependent protein C gi gi gi Hemoglobin beta chain gi beta embryonic-2 gi beta-2 subunit gi embryonic subunit gi alpha subunit alpha-1 gi subunit alpha-2 c gi subunit alpha-a gi subunit beta d gi gi gi gi gi subunit beta-1 gi gi alpha 2 Macroglobulin gi gi gi gi gi gi alpha 2-1 gi alpha-2-p gi Transferrins serotransferrin gi S 14

15 transferrin gi gi gi Proteins with White Blood Cell Functions Complement proteins C1qB gi C1qC gi C1q protein 4 gi C1r/s-A isotype gi C1s subcomponent gi C3 gi gi gi gi gi gi gi gi gi gi gi gi gi gi gi gi C3 isoform X1 gi gi C3-2 gi C3-2 precursor gi C4 gi gi C4-B gi C5 gi gi gi gi C6 gi gi gi gi C7 gi gi gi gi gi C8 alpha gi gi C8 beta gi gi gi gi gi C8 gamma chain isoform X1 gi S 15

16 C9 gi gi gi gi gi pro-c3-1 gi factor B gi gi factor H gi factor I gi Immunoglobulins delta heavy chain gi gi domain-containing receptor 1 gamma heavy chain variable region heavy chain gi gi gi gi gi heavy chain BrE-3 - gi mouse (fragment) heavy chain precursor gi heavy chain variable region kappa chain V region Mem5 kappa chain V-II region RPMI 6410 precursor protein gi gi gi gi gi gi gi gi gi gi kappa chain V-IV gi region JI precursor kappa light chain gi lambda light chain VLJ gi region lambda polypeptide 1 gi precursor light chain gi gi gi gi gi gi S 16

17 gi gi gi light chain isotype 1 gi light chain isotype L1 gi light chain precursor gi light chain precursor L gi light chain type 1 gi M heavy chain gi mu heavy chain gi gi gi gi gi gi mu heavy chain gi secretory form mu heavy chain gi variable region mu/tau heavy chain gi gi tau heavy chain gi VTG Female-specific Egg Proteins gi gi gi gi gi VTG A gi VTG Aa gi VTG Ab gi VTG C gi gi VTG-1 gi gi gi gi ZP glycoprotein 2.3 precursor gi Other Significant Proteins angiotensinogen gi angiotensinogen precursor gi S 17

18 CaMKIIa subunit alpha gi isoform 1 CaMKIIa subunit delta gi ceruloplasmin gi gi gi gi Ceruloplasmin isoform gi gi X1 VEGF chain A gi 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