In this report, first use of size-selected gold nanoparticles (AuNPs) as matrixes for matrix assisted laser desorption/ionization (MALDI) is described for peptides and proteins. In comparison with conventional organic acid MALDI matrixes, the optimum matrix-to-analyte ratio with AuNP matrixes is reduced by 10-14 orders of magnitude. Significant differences in the relative abundances of the ions observed in positive and negative mode MALDI-time-of-flight mass spectrometry (TOFMS) are revealed as the AuNP size distribution is decreased from 10 to 2 nm, whereby 2-nm AuNPs exhibit quantum confinement effects prevalent in quantum dots. AuNP matrixes allow for selective analyte ionization, as demonstrated in the selective MALDI-TOFMS of phosphotyrosine in a background of phosphoserine and phosphothreonine peptides.

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The rate of protein digestion imposes significant limitations on high-throughput protein identification using mass spectrometry. In this report, we demonstrate that proteins are readily digested by trypsin in the presence of organic solvents such as methanol, acetone, 2-propanol, and acetonitrile. The rates of protein digestion in organic solvents, as indicated by the abundances of digest fragment ions in the mass spectrum, are increased relative to aqueous solution. In addition, amino acid coverage for the analyzed proteins increases in the presence of the organic solvents, and proteins that are resistant to proteolysis are readily digested. For example, a 68% amino acid sequence coverage was attained from a tryptic digest of myoglobin in < 5 min from an 80% acetonitrile solution, whereas no digest fragments were detected from a 5 min digestion in an aqueous solution. Moreover, the tryptic digestion of a complex protein mixture in an organic-aqueous solvent system showed significantly enhanced digestion for nearly all of the protein components. Enzymatic digestion in an organic-aqueous solvent system is a rapid, simple, and effective peptide mass-mapping technique.

Ion mobility and mass spectrometry measurements have been used to examine the populations of different solution structures of the nonapeptide bradykinin. Over the range of solution compositions studied, from 0:100 to 100:0 methanol:water and 0:100 to 90:10 dioxane:water, evidence for ten independent populations of bradykinin structures in solution is found. In some solutions as many as eight structures may coexist. The solution populations are substantially different than the gas-phase equilibrium distribution of ions, which exhibits only three distinct states. Such a large number of coexisting structures explains the inability of traditional methods of characterization such as nuclear magnetic resonance spectroscopy and crystallography to determine detailed structural features for some regions of this peptide.

Neuropeptides are important messenger molecules that influence nearly all physiological processes. In insects, they can be released as neuromodulators within the central nervous system (CNS) or as neurohormones into the hemolymph. We analyzed the peptidome of neurohormonal release sites and associated secretory peptidergic neurons of adult Drosophila melanogaster. MALDI-TOF mass spectrometric analyzes were performed on single organs or cell cluster from individual flies. This first peptidomic characterization in adult fruit flies revealed 32 different neuropeptides. Peptides not directly predictable from previously cloned or annotated precursor genes were sequenced by tandem mass spectrometry. These peptides turned out to be either intermediate products of neuropeptide processing or shorter versions of known peptides. We found that the peptidome of the CNS-associated neurohemal organs is tagma-specific in Drosophila. Abdominal neurohemal organs and their supplying peptidergic neurons contain the capa gene products periviscerokinins and pyrokinin-1, thoracic neurohemal organs contain FMRFamides, and the neurohemal release sites of the brain contain pyrokinin-1(2-15), pyrokinin-2, corazonin, myosuppressin, and sNPF as their major putative release products. Our results show that peptidomic approaches are well suited to study differential neuropeptide expression or posttranslational modifications in morphologically defined parts of the nervous system and in a developmental and physiological context in animals as small as Drosophila melanogaster.