US Food and Drug Administration Perspectives on Clinical Mass Spectrometry.

Mass spectrometry-based in vitro diagnostic devices that measure proteins and peptides are underutilized in clinical practice, and none has been cleared or approved by the Food and Drug Administration (FDA) for marketing or for use in clinical trials. One way to increase their utilization is through enhanced interactions between the FDA and the clinical mass spectrometry community to improve the validation and regulatory review of these devices. As a reference point from which to develop these interactions, this article surveys the FDA's regulation of mass spectrometry-based devices, explains how the FDA uses guidance documents and standards in the review process, and describes the FDA's previous outreach to stakeholders. Here we also discuss how further communication and collaboration with the clinical mass spectrometry communities can identify opportunities for the FDA to provide help in the development of mass spectrometry-based devices and enhance their entry into the clinic.

Rarity gives a charm: evaluation of trace proteins in plasma and serum.

Since plasma potentially contacts every cell as it circulates through the body, it may carry clues both to diagnosis and treatment of disease. It is commonly expected that the growing ability to detect and characterize trace proteins will result in discovery of novel therapeutics and biomarkers; however, the familiar, super-abundant plasma proteins remain a fundamental stumbling block. Furthermore, robust validation of proteomic data is a sometimes overlooked but always necessary component for the eventual development of clinical reagents. This review surveys some of the uses of typical and atypical low-abundance proteins, current analytical methods, existing impediments to discovery, and some innovations that are overcoming the challenges to evaluation of trace proteins in plasma and serum.

Therapeutic potential of the plasma proteome.

Plasma contains numerous and diverse proteins with existing and potential therapeutic value. Plasma has been used clinically as both a source of purified derivatives for treating diseases such as hemophilia, and as a diagnostic medium. Recent research directed towards mining plasma's true potential takes advantage of state-of-the-art proteomic analytical methods to develop multi-protein, disease-specific biomarker panels to improve the reliability and specificity of diagnostics. Recombinant production and chromatographic purification methods are increasing the yield and safety of traditional plasma derivatives. Emerging cell-based technologies are being applied to discover novel protein activities and identify epitope-specific antibodies that may have clinical promise.

The Bead blot: a method for selecting small molecule ligands for protein capture and purification.

We present a new method for selecting peptide ligands that are useful for protein purification, protein targeting and exploring protein-ligand interactions, and which requires no prior protein purification or derivatization. In the Bead blot, a complex mixture containing the target protein, for example, plasma, is incubated with a combinatorial library of peptide ligands synthesized on beads. The proteins are fractionated and purified on their respective ligands and the beads with their bound proteins are immobilized in a gel. The proteins are eluted from the ligands by capillary action and captured on a membrane so that their position on the membrane corresponds to the position of the beads in the gel. The protein is detected on the membrane, generating spots on an autoradiography film, the spots on the film are aligned with the beads in the gel, and the beads that bound the protein are recovered. The ligand on the bead(s) can be sequenced and synthesized at large scale for protein purification. The Bead blot can be completed in several hours with overnight pause steps if desired. On average, 5 prospective ligands are selected per 50,000 beads screened using this method. Unlike other ligand identification methods, the target protein does not have to be purified or labeled, and the Bead blot allows ligands for multiple proteins to be selected in a single experiment.

The Bead blot: a method for identifying ligand-protein and protein-protein interactions using combinatorial libraries of peptide ligands.

Small molecules that bind proteins can be used as ligands for protein purification and for investigating protein-protein and protein-drug interactions. Unfortunately, many methods used to identify new ligands to desired proteins suffer from common shortcomings, including the requirement that the target protein be purified and/or the requirement that the ligands be selected under conditions different from those under which it will be used. We have developed a new method called the Bead blot that can (i) select ligands to unpurified proteins, including trace proteins, present in complex materials (e.g., unfractionated plasma); (ii) select ligands to multiple proteins under a variety of conditions in a single experiment; and (iii) be used with libraries of different types of ligands. In the Bead blot, a library of ligands, synthesized on chromatography resin beads, is incubated with a starting material containing a target protein for which a ligand is sought. The proteins in the material bind to their complementary ligands according to specific affinity interactions. Then the protein-loaded beads are immobilized in a porous matrix, and the proteins are directionally eluted from the beads and captured on a membrane superimposed on the beads. The location of the target protein on the membrane is determined, and because the position of the protein(s) on the membrane reflects the position of the bead(s) in the matrix, the bead that originally bound the protein is identified, with subsequent elucidation of the ligand sequence. Ligands to several targets can be identified in one experiment. Here we demonstrate the broad utility of this method by the selection of ligands that purify plasma protein complexes or that remove pathogens from whole blood with very high affinity constants. We also select ligands to a protein based on competitive elution.

Functional identification of novel activities: activity-based selection of proteins from complete proteomes.

The identification of proteins with desired activities, especially from complex samples such as plasma and whole blood, is a continual challenge. We have developed a technology platform called Functional Identification of Novel Activities (FIoNA) to discover desired protein activities from complex biological samples. FIoNA uses immobilized libraries of combinatorial peptide ligands to purify and concentrate essentially all of the components of a complex mixture on ligands synthesized on individual beads. No depletion or prefractionation of the starting material is performed before it is incubated with the library, and no a priori knowledge of the active protein or of the ligand to which it binds is required. Instead, the protein-loaded beads are individually evaluated en masse in disease- relevant assays to identify proteins possessing a desired function. Beads associated with the activity are selected, and the ligand is sequenced and resynthesized in bulk on the original backbone for purification and characterization of the active component. Here we illustrate the use of FIoNA in a cell proliferation assay to detect a growth factor present in conditioned cell medium at nanogram/milliliter concentrations. We also have selected beads associated with hydrolysis of nerve agent analogs in assays performed in 100,000-well microtiter plates.

Treatment of experimental osteomyelitis with a fibrin sealant antibiotic implant.

Two methods currently are available for the delivery of antibiotics: intravenous injection with a long-term indwelling catheter and local implant of antibiotic-containing polymethylmethacrylate beads. Both of these methods have significant disadvantages. A fibrin sealant implant, impregnated with tobramycin, was evaluated in a rabbit model of osteomyelitis to determine whether it has the potential of supplying a basis for bone reconstruction and providing an improved treatment method for the delivery of antibiotics to orthopaedic infections. Localized tibial osteomyelitis, with methicillin-sensitive Staphylococcus aureus, was developed surgically in female New Zealand White rabbits. After 2 weeks, rabbits with evidence of osteomyelitis were treated with debridement alone, debridement plus systemic tobramycin, debridement plus fibrin sealant, debridement plus fibrin sealant loaded with tobramycin, polymethylmethacrylate beads loaded with tobramycin, or not treated at all (control). After 4 weeks of therapy, the rabbits were sacrificed and the involved bones were cultured for concentrations of methicillin-sensitive Staphylococcus aureus per gram of bone and marrow. Preliminary data (N = 14) indicate fibrin sealant plus tobramycin may be as effective as polymethylmethacrylate beads plus tobramycin against methicillin-sensitive Staphylococcus aureus osteomyelitis in a rabbit model.