Journal of the American Chemical Society ARTICLE Figure 8. (A) Bait chemical structure determines aﬃnity and can capture or exclude select proteins. Poly(NIPAm/DB3) and poly- (NIPAm/RBB) nanoparticles were incubated with troponin-I. DB3 and RBB are anthraquinone rings that diﬀer in their side groups. RBB depleted greater than 99.9% (estimated dissociation constant KD < 10 11 M) while DB3 depleted 83% (KD = 10 10 M), at equilibrium (<10 min). (B) Proteins sequestered in the nanoparticles are protected from enzymatic degradation. Interleukin-8 (IL8) diluted in artiﬁcial sweat was incubated with excess trypsin at 37 C in the presence or absence of poly(NIPAm/DY9) particles. SDS PAGE analysis revealed that in the absence of poly(NIPAm/DY9) particles, the IL8 band disappeared, and a fainter band at lower molecular weight appeared, indicating the presence of a degradation product (lane 2). In the presence of poly- (NIPAm/DY9) particles, IL8 is fully protected from trypsin degradation (lane 5). Incubation of IL8 with sweat alone at 37 C caused the intact IL8 band to disappear (lane 3), while poly(NIPAm/DY9) particles yielded a full density band for the intact protein (lane 4), suggesting that IL8 was protected from degradation at 37 C by the nanoparticles. Figure 9. Poly(NIPAm/RB) particles eﬀectively increase the detection sensitivity for MRM analysis of human growth hormone (hGH) spiked in human urine. capture of all low-abundance ligands, (b) high-aﬃnity binding can protect captured proteins from degradation19 (Figure 8B), and (c) targeted low-molecular weight proteins bound to abun- dant carrier proteins in biological ﬂuid can be competed away by the high-aﬃnity bait and stripped away from the carrier protein.19 As shown in Figure 9, when poly(NIPAm/RBB) nanoparticles are introduced into a 1 mL sample containing 50 ng/mL of hGH, all of the hGH (50 ng) is sequestered in the nanoparticles in one step and can be eluted in 50 μL, yielding a 20-fold increase in analyte concentration to meet the MRM limits of sensitivity. A larger starting sample volume will yield a proportionally higher increase in analyte concentration. Figure 10. (A) Poly(NIPAm-co-VSA) particles excluded albumin while capturing interleukin 6 (IL6). SDS PAGE analysis shows that all the albumin is left in the supernatant (lane 2) while all the IL6 is captured by the particles (lane 3). For comparison purposes, poly(NIPAm/CB) nonshelled particles are shown. In this case all IL6 was captured (lane 4), but albumin was also detectable in the particles (lane 5) because CB binds to albumin species. (B) The addition of a vinylsulfonic acid (VSA)- functionalized shell to cibacron blue F3GA (CB)-containing particles decreased the number of unwanted albumin peptide hits (upper) while at the same time increased the frequency of MS hits for target proteins (e.g., Apo-CI) (lower) (Supporting Information Table 4). (C) Poly- (NIPAm/CB) core (NIPAm-co-VSA) shell particles greatly enhance the low-molecular weight protein concentration in whole blood (lane 3) while fully excluding high-molecular weight abundant proteins such as albumin and immunoglobulins. Our results suggest that particles containing organic struc- tures, such as vinyl sulfonic acid (VSA), excluded (1) full-length albumin by size sieving and (2) peptides derived from albumin that are below the size cutoﬀ of the eﬀective pore size of the particle by selective repulsion. VSA-containing particles excluded albumin but still retained aﬃnity for wide classes of other proteins and peptides. These ﬁndings are consistent with pre- vious literature in which VSA-containing polymers were selec- tively able to prevent albumin absorption49 but retained the ability to form complexes with proteins such as trypsin.50 Poly(NIPAm-co-VSA) particles completely excluded full- length bovine serum albumin (BSA) while sequestering inter- leukin-6 from solution (Figure 10A). In a separate experiment, fragment peptides derived from trypsinization of albumin were incubated with poly(NIPAm-co-VSA) particles. Albumin-de- rived peptides below the size exclusion cutoﬀ for the poly(NIP- Am-co-VSA) particles (Figure 5) were consistently excluded by the poly(NIPAm-co-VSA) particles at a variety of pH values (Table 2). These same particles that excluded low-molecular weight albumin fragments continued to sequester a broad variety 19184 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188