Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults
Abstract
:1. Introduction
2. Results
2.1. Characterization of Plasma Exosomes from Neonates and Adults
2.2. Differential Protein Expression
2.3. Biological Pathway Analysis
2.4. Validation of Differential Protein Expression in Independent Samples
3. Discussion
4. Materials and Methods
4.1. Sample Collection
4.2. Platelet-Free Plasma Isolation
4.3. Plasma Exosomes Isolation and Characterization
4.4. Quantitative Proteomic Studies by Liquid Chromatography Mass Spectrometry (LC-MS/MS) Using Sequential Window Acquisition of All Theoretical Mass Spectrometry (SWATH MS) Method
4.4.1. Protein Digestion
4.4.2. Mass Spectrometric Analysis by Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH MS)
4.5. Functional Enrichment and Interaction Network Analysis
4.6. Von Willebrand Factor and Protein S Determination
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
A2MG | α2-macroglobulin |
C4BP | C4b-binding protein |
CB | Cord blood |
DDA | Data-dependent acquisition |
DLS | Dynamic light scattering |
ELISA | Enzyme-linked immunosorbent assay |
FDR | False discovery rate |
FV and FVIII | Factor VIII and Factor V |
GNAI2 | Guanine nucleotide-binding protein G(i) subunit α-2 |
MVBs | Multivesicular bodies |
PBS | Phosphate-buffered saline |
PCA | Principal components analysis |
PF4 | Platelet factor 4 |
RAB11B | Ras-related protein Rab-11B |
RAP1A | Ras-related protein Rap-1A |
SCFD2 | Sec 1 family domain containing 2 |
SNARE | Soluble N-ethylmaleimide-sensitive fusion Attachment protein (SNAP) REceptors |
TEM | Transmission electron microscopy |
TLN1 | Talin-1 |
TSG-101 | Tumor susceptibility gene 101 protein |
vWF | von Willebrand factor |
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Uniprot Code | Gene Name | Protein Name | p-Value | FCh |
---|---|---|---|---|
P02786 | TFR1 | Transferrin receptor protein 1 | 0.00104 | 15.32 |
Q99808 | S29A1 | Equilibrative nucleoside transporter 1 | 0.01424 | 14.01 |
P11166 | GTR1 | Solute carrier family 2, facilitated glucose transporter member 1 | 0.00106 | 10.15 |
P69892 | HBG2 | Hemoglobin subunit γ-2 | 0 | 9.43 |
P02792 | FRIL | Ferritin light chain | 0.00438 | 9.22 |
Q16819 | MEP1A | Meprin A subunit α | 0.01481 | 7.13 |
P02776 | PLF4 | Platelet factor 4 | 0.00809 | 7.01 |
Q02094 | RHAG | Ammonium transporter Rh type A | 0.00772 | 6.71 |
P27918 | PROP | Properdin | 0 | 6.26 |
P16671 | CD36 | Platelet glycoprotein 4 | 0.00001 | 5.99 |
Q9NQ84 | GPC5C | G-protein-coupled receptor family C group 5 member C | 0.00467 | 5.87 |
P08195 | 4F2 | 4F2 cell surface antigen heavy chain | 0.00224 | 5.77 |
Q658P3 | STEA3 | Metalloreductase STEAP3 | 0.00262 | 5.34 |
Q6UX06 | OLFM4 | Olfactomedin-4 | 0.00008 | 5.08 |
P02787 | TRFE | Serotransferrin | 0.00101 | 5.05 |
P08514 | ITA2B | Integrin αIIb | 0 | 4.88 |
P02730 | B3AT | Band 3 anion transport protein | 0.00334 | 4.71 |
P11142 | HSP7C | Heat shock cognate 71 kDa protein | 0.00239 | 4.7 |
Q15758 | AAAT | Neutral amino acid transporter B(0) | 0.00829 | 4.56 |
P05164 | PERM | Myeloperoxidase | 0.00003 | 4.46 |
P0DMV9 | HS71B | Heat shock 70 kDa protein 1B | 0.0012 | 4.36 |
P27105 | STOM | Erythrocyte band 7 integral membrane protein | 0.00326 | 4.18 |
P02743 | SAMP | Serum amyloid P-component | 0 | 4.15 |
Q13201 | MMRN1 | Multimerin-1 | 0.00228 | 4.1 |
Q15907 | RB11B | Ras-related protein Rab-11B | 0.00058 | 4.03 |
Q9HD89 | RETN | Resistin | 0.00002 | 3.79 |
P01024 | CO3 | Complement C3 | 0 | 3.6 |
Q86UX7 | URP2 | Fermitin family homolog 3 | 0 | 3.51 |
P05023 | AT1A1 | Sodium/potassium-transporting ATPase subunit α-1 | 0.00875 | 3.39 |
P18428 | LBP | Lipopolysaccharide-binding protein | 0.00971 | 3.31 |
P04275 | VWF | von Willebrand factor | 0 | 3.29 |
Q00610 | CLH1 | Clathrin heavy chain 1 | 0.00667 | 3.15 |
P01859 | IGHG2 | Ig heavy constant γ 2 | 0.0001 | 3.06 |
Q9Y490 | TLN1 | Talin-1 | 0.00002 | 3.02 |
P00451 | FA8 | Coagulation factor VIII | 0.00055 | 3.01 |
P05106 | ITB3 | Integrin β3 | 0.00003 | 3 |
P01042 | KNG1 | Kininogen-1 | 0.00652 | 2.95 |
O15400 | STX7 | Syntaxin-7 | 0.00007 | 2.89 |
P12259 | FA5 | Coagulation factor V | 0 | 2.77 |
P26038 | MOES | Moesin | 0.00065 | 2.74 |
P10643 | CO7 | Complement component C7 | 0 | 2.73 |
Q9UP52 | TFR2 | Transferrin receptor protein 2 | 0.03685 | 2.71 |
P02760 | AMBP | Protein AMBP | 0.00118 | 2.7 |
P11597 | CETP | Cholesterylester transfer protein | 0.00012 | 2.7 |
P15144 | AMPN | Aminopeptidase N | 0.00896 | 2.6 |
P21926 | CD9 | CD9 antigen | 0.00006 | 2.59 |
P01861 | IGHG4 | Ig heavy constant γ 4 | 0.00009 | 2.58 |
P0C0L5 | CO4B | Complement C4-B | 0 | 2.54 |
Q71DI3 | H32 | Histone H3.2 | 0.0305 | 2.49 |
P05546 | HEP2 | Heparin cofactor 2 | 0.00001 | 2.44 |
P08603 | CFAH | Complement factor H | 0.0057 | 2.43 |
P13671 | CO6 | Complement component C6 | 0.00044 | 2.33 |
P04004 | VTNC | Vitronectin | 0 | 2.3 |
Q9HAV0 | GBB4 | Guanine nucleotide-binding protein subunit β-4 | 0.00045 | 2.27 |
P21333 | FLNA | Filamin-A | 0.00019 | 2.23 |
P02788 | TRFL | Lactotransferrin | 0.01132 | 2.23 |
P62834 | RAP1A | Ras-related protein Rap-1A | 0.00141 | 2.2 |
P01717 | LV325 | Ig λ variable 3-25 | 0.04177 | 2.18 |
P02675 | FIBB | Fibrinogen β chain | 0.00005 | 2.16 |
P69905 | HBA | Hemoglobin subunit α | 0.00317 | 2.15 |
P63104 | 1433Z | 14-3-3 protein ζ/δ | 0.00005 | 2.14 |
P02679 | FIBG | Fibrinogen γ chain | 0 | 2.13 |
P01857 | IGHG1 | Ig heavy constant γ 1 | 0.00004 | 2.1 |
P01023 | A2MG | A-2-macroglobulin | 0.00104 | 2.02 |
P06312 | KV401 | Ig κ variable 4–1 | 0.00234 | 0.48 |
P06310 | KV230 | Ig κ variable 2–30 | 0.00695 | 0.46 |
P07225 | PROS | Vitamin K-dependent protein S | 0.0011 | 0.42 |
P49721 | PSB2 | Proteasome subunit β type-2 | 0.01941 | 0.4 |
P01834 | IGKC | Ig κ constant | 0.00001 | 0.4 |
P01766 | HV313 | Ig heavy variable 3–13 | 0.00043 | 0.4 |
P0DOY3 | IGLC3 | Ig λ constant 3 | 0 | 0.39 |
A0A0C4DH69 | KV109 | Ig κ variable 1–9 | 0.00017 | 0.39 |
P04430 | KV116 | Ig κ variable 1–16 | 0.00037 | 0.37 |
B9A064 | IGLL5 | Ig λ-like polypeptide 5 | 0.00002 | 0.36 |
P27169 | PON1 | Serum paraoxonase/arylesterase 1 | 0.00304 | 0.36 |
Q93050 | VPP1 | V-type proton ATPase 116 kDa subunit a isoform 1 | 0.01287 | 0.35 |
P01700 | LV147 | Ig λ variable 1–47 | 0 | 0.35 |
P00338 | LDHA | L-lactate dehydrogenase A chain | 0.01646 | 0.34 |
P04003 | C4BPA | C4b-binding protein α chain | 0 | 0.33 |
P01601 | KVD16 | Ig κ variable 1D–16 | 0.00931 | 0.32 |
P02766 | TTHY | Transthyretin | 0.01393 | 0.32 |
Q6NSI8 | K1841 | Uncharacterized protein KIAA1841 | 0.04514 | 0.31 |
Q16851 | UGPA | UTP-glucose-1-phosphate uridylyltransferase | 0.04304 | 0.31 |
A0A0B4J1X5 | HV374 | Ig heavy variable 3–74 | 0.00052 | 0.31 |
A0A0C4DH25 | KVD20 | Ig κ variable 3D–20 | 0 | 0.31 |
P0DP03 | HV335 | Ig heavy variable 3–30–5 | 0 | 0.3 |
A0A0C4DH24 | KV621 | Ig κ variable 6–21 | 0.00154 | 0.28 |
P08519 | APOA | Apolipoprotein(a) | 0.00237 | 0.28 |
A2NJV5 | KV229 | Ig κ variable 2–29 | 0.00394 | 0.27 |
A0A0A0MS15 | HV349 | Ig heavy variable 3–49 | 0 | 0.27 |
A0A0C4DH68 | KV224 | Ig κ variable 2–24 | 0.00088 | 0.26 |
P01624 | KV315 | Ig κ variable 3–15 | 0.00002 | 0.25 |
P28072 | PSB6 | Proteasome subunit β type-6 | 0.02057 | 0.25 |
P04433 | KV311 | Ig κ variable 3–11 | 0.00002 | 0.24 |
P01591 | IGJ | Ig J chain | 0.00001 | 0.23 |
O43866 | CD5L | CD5 antigen-like | 0.00001 | 0.2 |
A0A0B4J1Y9 | HV372 | Ig heavy variable 3–72 | 0.00097 | 0.17 |
A0A075B6H9 | LV469 | Ig λ variable 4–69 | 0.00042 | 0.15 |
P00738 | HPT | Haptoglobin | 0.00134 | 0.13 |
P00739 | HPTR | Haptoglobin-related protein | 0 | 0.11 |
O14791 | APOL1 | Apolipoprotein L1 | 0.00002 | 0.08 |
P01877 | IGHA2 | Ig heavy constant α 2 | 0.00001 | 0.05 |
Q8TF72 | SHRM3 | Protein Shroom 3 | 0.01003 | 0.05 |
P01876 | IGHA1 | Ig heavy constant α 1 | 0.00001 | 0.02 |
Upregulated in Neonates vs. Adults | |||
Pathway | Protein Count | p Value | Proteins |
Hemostasis | 23 | <0.001 | RAP1A; YWHAZ; KNG1; FLNA; GNAI2; ITGA2B; ITGB3; A2MG; F5; PF4; PFN1; HIST2H3A; TLN1; FGA; FGB; F8; HIST2H3C; HIST2H3D; TF; FGG; CD36; vWF; HBG2 |
Formation of fibrin clot (clotting cascade) | 9 | <0.001 | KNG1; A2MG; F5; PF4; FGA; FGB; F8; vWF; FGG; |
Integrin cell surface interactions | 9 | <0.001 | RAP1A; ITGA2B; ITGB3; THBS1; VTN; TLN1; FGB; FGG; FGA; |
Complement cascade | 6 | <0.001 | MASP1; C3; C1R; C7; C6; C4B; |
β3 integrin cell surface interactions | 7 | <0.001 | ITGA2B; ITGB3; THBS1; VTN; FGA; FGB; FGG; |
Common pathway | 5 | <0.001 | F5; PF4; FGA; FGB; FGG; |
Platelet activation, signaling and aggregation | 10 | <0.001 | RAP1A; YWHAZ; FLNA; GNAI2; ITGA2B; ITGB3; PFN1; vWF; TF; TLN1; |
Ephrin B-EPHB pathway | 7 | =0.002 | RAP1A; ITGA2B; ITGB3; FGA; FGB; TF; FGG; |
p130Cas linkage to MAPK signaling for integrins | 4 | =0.002 | RAP1A; ITGA2B; ITGB3; TLN1; |
GRB2: SOS provides linkage to MAPK signaling for integrins | 4 | =0.002 | RAP1A; ITGA2B; ITGB3; TLN1; |
β2 integrin cell surface interactions | 5 | =0.008 | KNG1; FGA; FGB; C3; FGG; |
Ephrin B reverse signaling | 5 | =0.011 | ITGA2B; ITGB3; FGA; FGB; FGG; |
Initial triggering of complement | 4 | =0.014 | MASP1; C3; C1R; C4B; |
Netrin-mediated repulsion signals | 4 | =0.014 | RAP1A; ITGA2B; ITGB3; TLN1; |
Recycling pathway of L1 | 5 | =0.016 | RAP1A; CLTC; ITGA2B; ITGB3; TLN1; |
Proteoglycan syndecan-mediated signaling events | 27 | =0.025 | RAP1A; YWHAZ; CLTC; KNG1; FLNA; HSPA1A; GNAI2; SLC3A2; HSPA8; CLU; ITGA2B; ITGB3; A2MG; THBS1; VTN; TLN1; FGA; FGB; HSPA1B; TFRC; TF; FGG; SLC2A1; SDCBP; LBP; STEAP3; HBG2 |
Intrinsic pathway | 4 | =0.03 | KNG1; A2MG; F8; vWF; |
Integrin αIIbβ3 signaling | 4 | =0.047 | RAP1A; ITGA2B; ITGB3; TLN1; |
Downregulated in Neonates vs. Adults | |||
Pathway | Protein Count | p value | Proteins |
Chylomicron-mediated lipid transport | 3 | =0.006 | APOA1; APOB; APOC3; |
Lipoprotein metabolism | 3 | =0.033 | APOA1; APOB; APOC3; |
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Peñas-Martínez, J.; Barrachina, M.N.; Cuenca-Zamora, E.J.; Luengo-Gil, G.; Bravo, S.B.; Caparrós-Pérez, E.; Teruel-Montoya, R.; Eliseo-Blanco, J.; Vicente, V.; García, Á.; et al. Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults. Int. J. Mol. Sci. 2021, 22, 1926. https://doi.org/10.3390/ijms22041926
Peñas-Martínez J, Barrachina MN, Cuenca-Zamora EJ, Luengo-Gil G, Bravo SB, Caparrós-Pérez E, Teruel-Montoya R, Eliseo-Blanco J, Vicente V, García Á, et al. Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults. International Journal of Molecular Sciences. 2021; 22(4):1926. https://doi.org/10.3390/ijms22041926
Chicago/Turabian StylePeñas-Martínez, Julia, María N. Barrachina, Ernesto José Cuenca-Zamora, Ginés Luengo-Gil, Susana Belén Bravo, Eva Caparrós-Pérez, Raúl Teruel-Montoya, José Eliseo-Blanco, Vicente Vicente, Ángel García, and et al. 2021. "Qualitative and Quantitative Comparison of Plasma Exosomes from Neonates and Adults" International Journal of Molecular Sciences 22, no. 4: 1926. https://doi.org/10.3390/ijms22041926