Current methodologies for protein quantitation include 2-dimensional gel electrophoresis techniques, metabolic labeling, and stable isotope labeling methods to name only a few. The current literature illustrates both pros and cons for each of the previously mentioned methodologies. Keeping with the teachings of William of Ockham, "with all things being equal the simplest solution tends to be correct", a simple LC/MS based methodology is presented that allows relative changes in abundance of proteins in highly complex mixtures to be determined. Utilizing a reproducible chromatographic separations system along with the high mass resolution and mass accuracy of an orthogonal time-of-flight mass spectrometer, the quantitative comparison of tens of thousands of ions emanating from identically prepared control and experimental samples can be made. Using this configuration, we can determine the change in relative abundance of a small number of ions between the two conditions solely by accurate mass and retention time. Employing standard operating procedures for both sample preparation and ESI-mass spectrometry, one typically obtains under 5 ppm mass precision and quantitative variations between 10 and 15%. The principal focus of this paper will demonstrate the quantitative aspects of the methodology and continue with a discussion of the associated, complementary qualitative capabilities.

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Dissociation of the amide bonds in a protonated peptide leads to N-terminal sequence fragments with cyclic structures and C-terminal sequence fragments with linear structures. The ionic fragments containing the N-terminus (bn) have been shown to be protonated oxazolones, whereas those containing the C-terminus (Yn) are protonated linear peptides. The coproduced neutral fragments are cyclic peptides from the N-terminus and linear peptides from the C-terminus. A likely determinant of these structural choices is the proton affinity (PA) of the described peptide segments. This study determines the PA values of such segments (Pep), i.e., cyclic and linear dipeptides and a relevant oxazolone, based on the dissociations of proton-bound dimers [Pep + Bi]H+ in which Bi is a reference base of known PA value (Cooks kinetic method). The dissociations are assessed at different internal energies to thereby obtain both proton affinities as well as entropies of protonation. For species with comparable amino acid composition, the proton affinity (and gas phase basicity) follows the order cyclic peptide << oxazolone approximately linear peptide. This ranking is consistent with dissociation of the protonated peptide via interconverting proton-bound complexes involving N-terminal oxazolone (O) or cyclopeptide (C) segments and C-terminal linear peptide segments (L), viz. O...H+...L reversible C...H+...L. N-terminal sequence ions (bn) are formed with oxazolone structures which can efficiently compete for the proton with the linear segments. On the other hand, N-terminal neutral fragments detach as cyclic peptides, with H+ now being retained by the more basic linear segment from the C-terminus to yield Yn.

The neutral species eliminated upon fragmentation of fast‐moving mass‐selected ions can be directly identified by collisional ionization and detection in neutral fragment reionization (NfR) mass spectra. Establishment of the identity of neutral fragments yields valuable insight into the decomposition mechanism of a precursor ion, as demonstrated for fullerene and alkali metal iodide cluster ions as well as metal ion adducts of amino acids. In addition, neutral fragment reionization also provides structural information that may not be available from the complementary ionic fragments alone; this is illustrated in the differentiation of isomeric mononucleotides. The parameters influencing the appearance of NfR spectra are discussed and the scope and general applicability of the method are briefly evaluated.

fThe extended rod-like Plasmodium falciparum circumsporozoite protein (CSP) is comprised of three primary domains: a charged N terminus that binds heparan sulfate proteoglycans, a central NANP repeat domain, and a C terminus containing a thrombospondin-like type I repeat (TSR) domain. Only the last two domains are incorporated in RTS,S, the leading malaria vaccine in phase 3 trials that, to date, protects about 50% of vaccinated children against clinical disease. A seroepidemiological study indicated that the N-terminal domain might improve the efficacy of a new CSP vaccine. Using a panel of CSP-specific monoclonal antibodies, well-characterized recombinant CSPs, label-free quantitative proteomics, and in vitro inhibition of sporozoite invasion, we show that native CSP is N-terminally processed in the mosquito host and undergoes a reversible conformational change to mask some epitopes in the N-and C-terminal domains until the sporozoite interacts with the liver hepatocyte. Our findings show the importance of understanding processing and the biophysical change in conformation, possibly due to a mechanical or molecular signal, and may aid in the development of a new CSP vaccine.T he development of a vaccine to aid in the control of malaria is critical, as Plasmodium falciparum has evolved resistance to all antimalarial drugs deployed so far, including artemisinin (1). The leading malaria vaccine (RTS,S), currently in phase 3 trials, contains a formulated virus-like particle that encompasses the central and carboxyl-terminal domains of the circumsporozoite protein (CSP) fused to the hepatitis B virus surface antigen (2) and protects approximately 30% to 50% of infants or children from clinical disease for a limited duration (3, 4). Naturally derived human antibodies against a portion of the N-terminal region, including region 1, are associated with a reduced risk of disease (5), providing a basis to design new CSP vaccines. This N-terminal region of the CSP is absent from RTS,S.The importance of understanding protein structure because of its impact on the induction of broadly neutralizing antibodies and subsequent vaccine design continues to be revealed in the HIV arena (6, 7). In malaria, the importance of protein conformation for the induction of neutralizing antibodies was recently shown in vivo for an orthologue of the leading asexual-stage malaria vaccine antigen apical membrane antigen-1 (AMA-1). Only a recombinant AMA-1 forming a stable complex with a constrained synthetic rhoptry neck protein-2 peptide induced protective antibodies in vivo against a lethal blood-stage challenge malaria parasite infection (8). When developing a novel CSP vaccine, these more recent developments need to be considered with regard to the potential for changes within the CSP, such as through in vivo processing or conformational changes (9, 10) in a protein with a known extended rod-like structure (11), that could mask the adhesion domains located at the N-and C-terminal domains (9).To address these questions, a panel of CSP-speci...