The exposure of electrospray droplets to acid vapors can significantly affect protein charge state distributions (CSDs) derived from unbuffered solutions. Such experiments have been conducted by leaking acidic vapors into the counter-current nitrogen drying gas of an electrospray interface. Based on changes in protein CSDs, protein folding and unfolding phenomena are implicated in these studies. Additionally, non-covalently-bound complexes are preserved and transient intermediates observed, such as high charge state ions of holomyoglobin. CSDs of proteins containing disulfide bonds shift slightly, if at all, with acid vapor leak-in, but when these disulfide bonds are reduced in solution, charge states higher than the number of basic sites (Lys, Arg, His and N-terminus) are observed. Since there is no observed change in the CSD of buffered proteins exposed to acidic vapors, this novel multiple charging phenomenon is attributed to a pH effect. Thus, this acid vapor leak-in approach can be used to reverse ‘wrong-way-round’ nanoelectrospray conditions by altering solution pH in the charged droplets relative to the pH in bulk solution. In general, the exposure of electrospray droplets to acidic vapors provides means for altering protein CSDs independent of bulk unbuffered solution pH.

The dissociation of model RNA anions has been studied as a function of anion charge state and excitation amplitude using ion trap collisional activation. Similar to DNA anions, the precursor ion charge state of an RNA anion plays an important role in directing the preferred dissociation channels. Generally, the complementary c/y-ions from 5= P-O bond cleavage dominate at low to intermediate charge states, while other backbone cleavages appear to a limited extent but increase in number and relative abundance at higher excitation energies. The competition between base loss, either as a neutral or as an anion, as well as the preference for the identity of the lost base are also observed to be charge-state dependent. To gain further insight into the partitioning of the dissociation products among the various possible channels, model dinucleotide anions have been subjected to a systematic study. In comparison to DNA, the 2=-OH group on RNA significantly facilitates the dissociation of the 5= P-O bond. However, the degree of excitation required for a 5= base loss and the subsequent 3= C-O bond cleavage are similar for the analogous RNA and DNA dinucleotides. Data collected for protonated dinucleotides, however, suggest that the 2=-OH group in RNA can stabilize the glycosidic bond of a protonated base. Therefore, base loss from low charge state oligonucleotide anions, in which protonation of one or more bases via intramolecular proton transfer can occur, may also be stabilized in RNA anions relative to corresponding DNA anions. t has been recognized that various noncoding RNA species play important roles in cellular function [1][2][3][4]. Recently, significant effort has been focused on the identification of these noncoding RNAs. However, conventional methods are usually labor intensive and ineffective in the characterization of RNA modifications [5]. On the other hand, tandem mass spectrometry is a rapid and sensitive alternative, provided the desired structural information can be derived from the dissociation chemistry that occurs between stages of mass analysis.To develop tandem mass spectrometry-based RNA sequencing methods, an understanding of the factors that determine the nature of the structural information that can be obtained via dissociation of RNA ions is necessary. Some of the first studies on RNA dissociation were conducted with ions generated from fast atom bombardment (FAB). A variety of fragment ions, including the characteristic structural fragment c x -ion, were identified [6,7]. The combination of matrixassisted laser desorption ionization (MALDI) and electrospray ionization (ESI) with tandem mass spectrometry facilitated the study of the gas-phase dissociation of larger oligonucleotide ions. However, far more attention has been dedicated to the study of the dissociation behavior of DNA ions than to dissociation of RNA ions [8]. Using MALDI combined with a quadrupole/timeof-flight (Q/TOF) tandem mass spectrometer, Kirpekar and Krogh [9], for example, investigated the dissociation behavior of singly...

Gas-phase ion/ion reactions are emerging as flexible means for probing and manipulating analyte ions with particular utility in bioanalysis.What are the elements of an analytically useful chemical reaction? This question is implicit in much of analytical chemistry but is rarely addressed explicitly. Nevertheless, sometimes it is useful to articulate the obvious in order to place into context the relative strengths and weaknesses of the "chemical" part of an analytical method. Any list of desirable reaction characteristics would include high reaction efficiencysideally, 100% conversion of a reactant of interest (usually the analyte) to product(s). This trait underlies titrimetry, for example. Another obvious characteristic on the list is formation of products that are readily distinguishable from the reactants by available methods. For example, the naked eye can detect the color change associated with an acid-base or redox indicator. Instrumental techniques are a much more sophisticated means for "seeing" differences between reactants and products via, for example, spectroscopic, electrochemical, chromatographic, or mass spectral properties. A third criterion is that the reaction must perform a desirable function, such as precipitating an analyte in solution or making a transformation that facilitates a subsequent step in the process. These three characteristics are either necessary or desirable for any chemical reaction used in analysis. A fourth characteristic of selectivity might also be included, although no generalizations can be made in this regard. In some cases, for example, assays based on selective catalysts, such as enzymes, benefit from a high degree of selectivity. In other cases, "universal" reactions are desired, at least within a class of compounds (e.g., in the conversion of organic nitrogen compounds to ammonia in a Kjeldahl determination).This report describes the emergence of a new class of analytically useful chemical reactionssviz., gas-phase ion/ion reactionssimplemented within the context of an MS experiment. The advent of ionization methods that can generate multiplycharged ions, principally ESI and its variants, 1 has made the exploration of this reaction type possible. Chemical reactions have always played key roles in organic and biological MS experiments when both mass and structural information are of interest. The formation of gaseous ions, a sine qua non for an MS experiment, generally involves both chemical and physical processes, the former often causing the removal or attachment of an electron or ion in the gas phase. Mass and structural information for a molecule of interest is obtained via measurement of the mass-tocharge ratios (m/z) of the ions derived from the molecule. In this sense, the gaseous ion serves as a surrogate for the molecule of interest. Structural information, which has largely been restricted to information about bond connectivity, has generally been derived from unimolecular dissociation of the ion. This fragmentation may result from energy deposited into t...

A tandem mass spectrometry approach is demonstrated for complete sequencing of a model small interfering RNA (siRNA) based on ion trap collisional activation of intact single-stranded anions. Various charge states of the siRNA duplex and the individual strands were generated by nanoelectrospray (nano-ESI). The siRNA duplex anions were predominantly dissociated into the sense and antisense strands by collisional activation. The characteristic fragment ions (c/y- and a-B/w-ion series) from both strands were observed when higher activation amplitude was applied and when beam-type collisional activation was examined; however, the coexistence of fragment ions from both strands complicated spectral interpretation. The effect of precursor ion charge state on the dissociation of the individual sense and antisense strand siRNA anions was studied using ion trap collision-induced dissociation under various activation amplitudes. Through the activation of relatively low charge state precursor ions at relatively low excitation energy, selective backbone dissociation predominantly via the c/y channels was achieved. By applying relatively high excitation energy, the a-B/w channels also became prominent; however, the increase in spectral complexity made complete peak assignment difficult. In order to simplify the product ion spectra, proton-transfer reactions were applied, and complete sequencing of each strand was achieved. The application of tandem mass spectrometry to intact single-stranded anions demonstrated in this study can be adapted for the rapid identification of other noncoding RNAs in RNomics studies.

The Cao Vit gibbon (Nomascus nasutus) was rediscovered in 2002 in Vietnam and then in 2006 in China. This is the only known population with about 110 individuals located along the China-Vietnam border. Little is known about it other than its population size and distribution. We describe the social structure and group dynamics of the Cao Vit gibbons in China based on 2 years of monitoring from 2007 to 2009. Four established study groups at this site consisted of 1 adult male, 2 adult females and 2–6 offspring. Two juveniles in 2 groups disappeared during the research. Four infants were born in 3 groups from November 2008 to February 2009. In 2 of the groups, both adult females had dependent infants. These observations suggest that Cao Vit gibbons live in polygynous groups, contrary to the usual monogamous group with only 1 adult female, but nevertheless similar to the social organization of both N. concolor and N. hainanus. We observed a coordinated dispersal of 1 adult male and 2 large juveniles, and the male formed a pair with a newly arrived female. Our observations support a growing awareness of variability in gibbon social organization.