Disease-associated blood biomarkers exist in exceedingly low concentrations within complex mixtures of high-abundance proteins such as albumin. We have introduced an affinity bait molecule into N-isopropylacrylamide to produce a particle that will perform three independent functions within minutes, in one step, in solution: a) molecular size sieving b) affinity capture of all solution phase target molecules, and c) complete protection of harvested proteins from enzymatic degradation. The captured analytes can be readily electroeluted for analysis.There is an urgent need to discover novel biomarkers that provide sensitive and specific disease detection1 , 2. Cancer is rapidly becoming the leading cause of death for many population groups in the United States, largely due to the fact that the disease is usually diagnosed after the cancer has metastasized and treatment is ineffective. It is widely believed that early detection of cancer prior to metastasis will lead to a dramatic improvement in treatment outcome. Biomarkers are nucleic acids, proteins, protein fragments or metabolites indicative of a specific biological state, that are associated with the risk of contraction or presence of disease3. Biomarker research has revealed that low-abundance circulating proteins and peptides present a rich source of information regarding the state of the organism as a whole 4 . Two major hurdles have prevented these discoveries from reaching clinical benefit: 1) disease-relevant biomarkers in blood or body fluids may exist in exceedingly low concentrations within a complex mixture of biomolecules and could be masked by high-abundance species such as albumin, and 2) degradation of protein biomarkers can occur immediately following the collection of blood or body fluid as a result of endogenous or exogenous proteinases. The goal of this study was to create "smart" nano-particles that allow enrichment and encapsulation of selected classes of proteins and peptides from complex mixtures of biomolecules such as plasma, and protect them from degradation during subsequent sample handling. The captured analytes can be readily extracted from the particles by electrophoresis allowing for subsequent quantitative analysis. This nanotechnology provides a powerful tool that is uniquely suited for the discovery of novel biomarkers for early stage diseases such as cancer.SUPPORTING INFORMATION AVAILABLE: Available in the Supplementary Information are details on particles synthesis protocol, SDS PAGE analysis on molecular sieving properties and enzymatic degradation, and tables (Table S1 and S2) listing proteins (with peptide coverage lists) identified via LC-MS/MS (ESI) on material electroeluted from NIPAm and NIPAm/AAc particles. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript Nano Lett. Author manuscript; available in PMC 2010 May 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe concentration of proteins and peptides comprising t...

BackgroundChronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their anti-microbial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms.ResultsThe helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective anti-microbial, anti-attachment as well as anti-biofilm activity at concentrations in the low μg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and anti-biofilm activities.ConclusionsThe NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and potential future topical therapeutics for treating chronic wound infections.

Unique intramolecular rearrangement product ions have been observed in the product ion spectra of a number of peptides. Multiple stages of mass analysis (MS n ), molecular modeling, and chemical modifications of peptides have been used to provide insight into the mechanism of this rearrangement reaction. The rearrangement process begins with a four-residue immonium ion that transfers a proton from the immonium nitrogen to the primary amine on the N-terminus. The proton transfer leads to the rearrangement of the peptide, exposing an internal amino acid on the terminus of the new ion. The internal amino acid that becomes the terminus of the new ion is then readily lost. The reaction seems to benefit from an extended experimental time frame available for reaction. The reaction is most prominent in quadrupole ion trap and Fourier-transform ion cyclotron resonance experiments, is observed under some conditions in a triple quadrupole, but is not seen in a sector instrument. Without previous knowledge of this process, the peptide sequence as determined by MS/MS could be misidentified.

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen and its ability to produce biofilms is a direct impediment to the healing of wounds and recovery from infection. Interest in anti-microbial peptides (AMPs) has grown due to their potential therapeutic applications and their possible use against antibiotic resistant bacteria. LL-37 is the only cathelicidin expressed by humans. In this study, we tested LL-37 and the effect of a protease-resistant LL-37 peptide mimetic, the peptide enantiomer D-LL-37, for anti-microbial and anti-biofilm activity against P. aeruginosa. Both forms of the peptide were equally effective as AMPs with similar killing kinetics. Circular dichroism spectra were obtained to demonstrate the chirality of D- and L-LL-37, and the trypsin resistance of D-LL-37 was confirmed. The helical cathelicidin from the cobra Naja atra (NA-CATH), and synthetic peptide variations (ATRA-1, ATRA-2, NA-CATH:ATRA1-ATRA1) were also tested. Although the cobra cathelicidin and related peptides had strong anti-microbial activity, those tested did not inhibit Pseudomonas biofilm formation, neither did control peptides. Both D- and L-LL-37 inhibited the attachment of Pseudomonas to a 96-well plate and decreased the amount of pre-formed (established) biofilm. D-LL-37 is able to promote Pseudomonas motility and decrease biofilm formation by altering the rate of twitching as well as by downregulating the expression of the biofilm-related genes, rhlA and rhlB, similar to L-LL-37. Both L- and D-LL-37 protected Galleria mellonella in vivo against Pseudomonas infection, while NA-CATH:ATRA1-ATRA1 peptide did not. This study demonstrates the ability and equivalence of D-LL-37 compared to L-LL-37 to promote bacterial twitching motility and inhibit biofilm formation, and protect against in vivo infection, and suggests that this peptide could be a critical advancement in the development of new treatments for P. aeruginosa infection.

BackgroundThe blood proteome is thought to represent a rich source of biomarkers for early stage disease detection. Nevertheless, three major challenges have hindered biomarker discovery: a) candidate biomarkers exist at extremely low concentrations in blood; b) high abundance resident proteins such as albumin mask the rare biomarkers; c) biomarkers are rapidly degraded by endogenous and exogenous proteinases.Methodology and Principal FindingsHydrogel nanoparticles created with a N-isopropylacrylamide based core (365 nm)-shell (167 nm) and functionalized with a charged based bait (acrylic acid) were studied as a technology for addressing all these biomarker discovery problems, in one step, in solution. These harvesting core-shell nanoparticles are designed to simultaneously conduct size exclusion and affinity chromatography in solution. Platelet derived growth factor (PDGF), a clinically relevant, highly labile, and very low abundance biomarker, was chosen as a model. PDGF, spiked in human serum, was completely sequestered from its carrier protein albumin, concentrated, and fully preserved, within minutes by the particles. Particle sequestered PDGF was fully protected from exogenously added tryptic degradation. When the nanoparticles were added to a 1 mL dilute solution of PDGF at non detectable levels (less than 20 picograms per mL) the concentration of the PDGF released from the polymeric matrix of the particles increased within the detection range of ELISA and mass spectrometry. Beyond PDGF, the sequestration and protection from degradation for a series of additional very low abundance and very labile cytokines were verified.Conclusions and SignificanceWe envision the application of harvesting core-shell nanoparticles to whole blood for concentration and immediate preservation of low abundance and labile analytes at the time of venipuncture.

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Urine is a potential source of diagnostic biomarkers for detection of diseases, and is a very attractive means of non-invasive biospecimen collection. Nonetheless, proteomic measurement in urine is very challenging because diagnostic biomarkers exist in very low concentration (usually below the sensitivity of common immunoassays) and may be subject to rapid degradation. Hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB) have been applied to address these challenges for urine biomarker measurement. We chose one of the most difficult low abundance, but medically relevant, hormones in the urine: human growth hormone (hGH). The normal range of hGH in serum is 1 to 10 ng/mL but the urine concentration is suspected to be a thousand times less, well below the detection limit (50 pg/mL) of sensitive clinical hGH immunoassays. We demonstrate that CB particles can capture, preserve and concentrate hGH in urine at physiological salt and urea concentrations, so that hGH can be measured in the linear range of a clinical immunometric assay. Recombinant and cadaveric hGH were captured from synthetic and human urine, concentrated and measured with an Immulite chemiluminescent immunoassay. Values of hGH less than 0.05 ng/mL (the Immulite detection limit) were concentrated to 2 ng/mL, with a urine volume of 1 mL. Dose response studies using 10 mL of urine demonstrated that the concentration of hGH in the particle eluate was linearly dependent on the concentration of hGH in the starting solution, and that all hGH was removed from solution. Thus if the starting urine volume is 100 mL, the detection limit will be 0.1 pg/mL. Urine from a healthy donor whose serum hGH concentration was 1.34 ng/mL was studied in order detect endogenous hGH. Starting from a volume of 33 mL, the particle eluate had an hGH concentration of 58 pg/mL, giving an estimated initial concentration of hGH in urine of 0.175 pg/mL. The nanotechnology described here appears to have the desired precision, accuracy and sensitivity to support large scale clinical studies of urine hGH levels. Nano Research503 Nano Res (2008) 1:502 518

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