Journal of the American Chemical Society Table 2. Example Peptides Derived from Human Serum Albumin That Were Excluded by Poly(NIPAm-co-VSA) Par- ticles at Diﬀerent pH Valuesa peptide hits (P/S) peptide pI MW pH 8 pH 4 pH 2 K.AEFAEVSK.L 6.5 1121.3 0/0 0/0 0/1 K.AVM*DDFAAFVEK.C 4.4 1653.8 0/22 6/30 0/0 K.AVMDDFAAFVEK.C 4.4 1573.8 0/51 8/48 0/0 K.DLGEENFK.A 4.4 1150.2 0/0 0/3 0/2 K.FQNALLVR.Y 10.4 1251.4 0/71 22/105 18/68 K.KLVAASQAALGL.- 10.8 1269.5 0/18 0/0 1/12 K.KQTALVELVK.H 10.5 1393.6 0/31 6/15 4/14 K.KYLYEIAR.R 10.4 1339.6 0/26 0/4 1/9 K.LDELRDEGK.A 4.5 1273.4 0/1 0/14 0/10 K.LVAASQAALGL.- 9.7 1141.3 0/76 12/120 10/66 K.PLVEEPQNLIK.Q 6.5 1535.8 0/4 0/3 0/1 K.SLHTLFGDK.L 9.5 1258.4 0/6 0/5 0/0 K.VPQVSTPTLVEVSR.N 9.7 1754.0 0/42 7/11 6/13 K.YLYEIAR.R 10.0 1211.4 0/1 15/11 11/32 R.FKDLGEENFK.A 6.6 1453.6 0/126 10/26 20/22 R.HPDYSVVLLLR.L 9.3 1580.6 0/0 2/1 0/2 R.RPCFSALEVDETYVPK.E 4.6 2139.4 0/0 0/2 0/0 a Albumin was trypsin digested before particle incubation. Poly(NIPAm- co-VSA) particles excluded all albumin tryptic peptides at pH 8. P = particle content, S = supernatant. of non-albumin peptides below the size exclusion cutoﬀ of the poly(NIPAm-co-VSA) particles (Figure 5 and Supporting In- formation Table 1). The mechanism by which poly(NIPAm-co- VSA) particles repel albumin-derived peptides is not completely known and cannot be explained by simple charge eﬀects. In fact, albumin peptides below their calculated isoelectric point at pH 8 (therefore carrying net positive charges) did not bind to the highly ionized sulfonate groups. Excluded peptides derived from albumin may not display the correct pattern of hydrophobic residues and charge distribution that matches the VSA-function- alized nanoparticle landscape. Exclusion of classes of peptides by speciﬁc bait chemistries is a new principle in peptide separation. In order to maximize the performance of the particles, we added a shell containing VSA to poly(NIPAm/CB) core parti- cles. CB is known to bind albumin and albumin peptides (along with a broad number of other proteins) with high aﬃnity. We performed MS analysis of poly(NIPAm/CB) core poly- (NIPAm-co-VSA) shell particle puriﬁed plasma. Albumin pep- tide hits present in the particle eluate dramatically decreased if the CB bait in the core was separated from the plasma by the VSA shell (Figure 10B). Since the molecular weight cutoﬀ of the particles is 30 kDa (Figure 10C) and albumin fragments are normally present in the blood,20 albumin species excluded by the particles are not only full-length molecules but also albumin fragments below 30 kDa. Thus, in one step, undesired albumin species are excluded. The addition of the VSA shell did not hinder the capture of other target analytes (e.g., Apo CI, Figure 10B). Poly(NIPAm/CB) core poly(NIPAm-co-VSA) shell particles allowed for the se- quencing of low-abundance, low-molecular weight plasma pro- teins (Table 3) that were not identiﬁed without the dual function nanoparticles. An example of 200 proteins that were captured by ARTICLE poly(NIPAm/CB) core poly(NIPAm-co-VSA) shell particles is shown in Supporting Information Table 3. Since the molec- ular weight cutoﬀ of poly(NIPAm/CB) core poly(NIPAm-co- VSA) shell particles is 30 kDa we can state that 65% of captured proteins was represented in serum as fragments of the parent molecule.51 We investigated the power and extent of protein and peptide enrichment for low-molecular weight, low-abundance proteins in a pool of healthy donor sera or plasma. We compared the peptides identiﬁed by LC MS/MS with and without a one-step processing through poly(NIPAm/CB) core poly(NIPAm-co- VSA) shell particles. A large number of diagnostic low-molecular weight proteins and peptides whose concentration in blood is less than 10 ng/mL were identiﬁed by MS after poly(NIPAm/CB) core poly- (NIPAm-co-VSA) shell nanoparticle harvesting. Moreover, an additional set of peptides belonging to low-abundance proteins were identiﬁed that were not previously found by MS analysis in whole blood, serum, or EDTA plasma, nor included in the PeptideAtlas database, the most comprehensive high conﬁdence collection of MS identiﬁed proteins52,53 (Supporting Informa- tion Figures 1 and 2 and Tables 1 and 2). Compared to the samples processed without nanoparticle harvesting, the ability of the nanoparticles to concentrate the low-abundance pepti- dome from whole blood was evident by SDS PAGE analysis (Figure 10C). Proteins whose molecular weight was lower than 30 kDa were captured by the particles. Thus, the nanoparticle technology described herein can be used for biomarker discovery in whole blood as a single preprocessing step. The nanoparticles will remain in the plasma following low-speed centrifugation to remove the cellular content (Figure 3). In the future it will be important to evaluate the eﬀect of potential interfering sub- stances present in whole blood and plasma such as lipemia, bilirubinemia, and hemolysis. Harvesting hydrogel nanoparticles possess important features, as we have demonstrated in previous studies using poly(NIPAm- co-AAc) and poly(NIPAm/CB): (a) use of the nanoparticles ampliﬁes the eﬀective sensitivity (by concentrating the sample analyte into a smaller volume and excluding unwanted contam- inants) while maintaining the linearity of quantitative im- munoassays;16 (b) nanoparticle preprocessing generated high yields and high precision when applied to clinical grade bio- marker studies;17 (c) if a quantitative immunoassay is previously established, nanoparticle preprocessing is highly suitable for veriﬁca- tion and validation of MS-identiﬁed candidate biomarkers.18 The new type of hydrogel core shell nanoparticles (poly- (NIPAm/CB) core poly(NIPAm-co-VSA) shell particles) de- scribed herein achieved a 10,000 fold eﬀective ampliﬁcation of the low-abundance, low-molecular weight proteins and peptides that could be identiﬁed by MS as demonstrated by examples of more than 200 low-abundance proteins (Supporting Information Table 3; Figure 11). The result was the identiﬁcation of a large series of novel candidate proteins not previously identiﬁed with mass spectrometry analysis in human serum (Table 3, Support- ing Information Tables 1 and 2 and Figure 1). While these candidate protein spectra have been manually validated (Support- ing Information Figure 2) and high stringency MS cutoﬀ ﬁlters have been applied (Experimental Section), a limitation of the current study is that completely new antibodies and quantitative immunoassays will have to be developed to independently verify these proteins by immunochemical methods in serum. 19185 dx.doi.org/10.1021/ja207515j |J. Am. Chem. Soc. 2011, 133, 19178–19188