Journal of the American Chemical Society ARTICLE Table 3. Example Candidate Proteins Identiﬁed by LC MS/MS LTQ Orbitrap Analysis for Samples Preprocessed with Poly(NIPAm/CB) Core Poly(NIPAm-co-VSA) Shell Nanoparticles That Were Not Present in Samples Preprocessed with Poly(NIPAm/CB) Unshelled Nanoparticles or Poly(NIPAm-co-VSA) Unshelled Particlesa accession protein blood concentration [pg/mL] and ref 38455402 lipocalin 2 (oncogene 24p3) [Homo sapiens] 5054 27765091 ST6 β-galactosamide α-2,6-sialyltranferase 1 isoform a [Homo sapiens] 15,00055 148664205 endothelial cell adhesion molecule [Homo sapiens] 30,00056 4759074 small inducible cytokine A16 precursor [Homo sapiens] 30,00057 4502101 annexin I [Homo sapiens] 50,00058 163659899 insulin-like growth factor 1 (somatomedin C) 100,00059 11321593 insulin-like growth factor binding protein 6 150,00059 62243290 insulin-like growth factor binding protein 4 400,00060 10834982 insulin-like growth factor binding protein 5 400,00060,61 144953895 nidogen 2 precursor [Homo sapiens] 12,000,00061 49574514 matrix Gla protein [Homo sapiens] 90,000,00062 33186925 AMP-activated protein kinase γ-2 subunit isoform a [Homo sapiens U 157168362 nucleoside phosphorylase [Homo sapiens] U 4507475 transglutaminase 1 [Homo sapiens] U 4502693 CD9 molecule [Homo sapiens] U 48375180 CD36 antigen [Homo sapiens] U 9257257 WD repeat domain 1 [Homo sapiens] U 222080092 WD repeat domain 91 [Homo sapiens] U 40254462 guanine nucleotide binding protein (G protein), q polypeptide [Homo sapiens] U a U = unknown. The presence of the VSA shell greatly increases the number and dynamic range of proteins sequestered for discovery. Figure 11. Poly(NIPAm/CB) core poly(NIPAm-co-VSA) shell na- noparticles increase the sensitivity of LC MS/MS analysis by 4 orders of magnitude as demonstrated by examples of more than 200 low- abundance proteins speciﬁed in Supporting Information Table 3. Conventional MS/MS analysis (90 min linear gradient at 500 nL/ min, data-dependent mode, Xcorr versus charge g1.9, 2.2, 3.5 for 1+, 2+, 3+ ions; ΔCn > 0.1; probability of randomized identiﬁcation e0.01) was used to compare the same serum set before and after nanoparticle processing. The ultimate sensitivity of the analysis depends on the starting sensitivity of the system. ’ CONCLUSIONS High buoyancy, polymer open-meshwork, core shell hydro- gel nanoparticles covalently functionalized with combinations of novel organic dye baits oﬀer a one-step technology for biomarker harvesting, concentration, and stabilization, with simultaneous exclusion of unwanted abundant molecules. The nanoparticles exhibit three physicochemical properties: (1) speciﬁc novel dye chemistries immobilized in the particle core possess extremely high aﬃnity for select classes of proteins and peptides; (2) a chemical species present in the shell, VSA, selectively excludes fragments and peptides derived from albumin, and (3) the outer shell of the particle performs physical size sieving, excluding large unwanted abundant molecules such as immunoglobulins. Dual- function hydrogel nanoparticles can dramatically magnify small protein and peptide identiﬁcation and measurement from complex biological solutions, using any current or future an- alytical platform, while stabilizing the analytes against degrada- tion. The ﬂuid samples used in the present sample were blood, urine, and sweat. The technology is applicable to any biologic solution-phase sample. We are currently incorporating nano- particles into a skin patch that continuously harvests, concen- trates, and preserves analytes present in the skin transudate for proteomic analysis. Bait-loaded core shell nanoparticles are being used for biomarker discovery in lymph, saliva, and cerebrospinal ﬂuid. ’ ASSOCIATED CONTENT bS Supporting Information. Experimental details including mass spectrometry analysis of serum, EDTA plasma, and whole blood preprocessed with the nanoparticles and complete refs 17 and 53. This material is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author aluchini@gmu.edu 19186 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188