Ultraviolet Photodissociation and Activated Electron Photodetachment Mass Spectrometry for Top-Down Sequencing of Modified Oligoribonucleotides.

With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'-O-methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'-O-methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.

The Role of Concentration Dependent Static Permittivity of Electrolyte Solutions in the Debye-Hückel Theory.

The Debye-Hückel theory has been extended to allow for arbitrary concentration dependence of the electrolyte solution static permittivity. The theory follows the lines advanced by Erich Hückel ( Hückel, E. Phys. Z. 1925, 26, 93) but gives rise to more general and lucid results. New theoretical expressions have been obtained for the excess free energy of solution, activity coefficient of water and mean ionic activity coefficient. The thermodynamic functions contain two terms representing interionic interactions and ion-water (solvation) interactions. The theory has been applied to calculate the activity coefficients of components in the aqueous solutions of alkali metal chlorides from LiCl to CsCl at ambient conditions making use of permittivities taken from experimental dielectric relaxation studies. Calculations without parameter adjustment have demonstrated a semiquantitative agreement with experimental data, reproducing both the nonmonotonic concentration dependence of the activity coefficients and the ordering of activity coefficients for the salts with different cations. A good agreement with experimental data is obtained for the aqueous solutions of LiCl in the concentration range up to 10 mol/kg. The nonmonotonic concentration dependence of activity coefficients is explained as a result of a balance between the effect of interionic interactions and the solvation contribution which appears quite naturally in the framework of the Debye-Hückel approach after incorporation of variable permittivity of solution.

Nonlinear fitting method for determining local false discovery rates from decoy database searches.

False discovery rate (FDR) analyses of protein and peptide identification results using decoy database searching conventionally report aggregate or global FDRs for a whole set of identifications, which are often not very informative about the error rates of individual members in the set. We describe a nonlinear curve fitting method for calculating the local FDR, which estimates the chance that an individual protein (or peptide) is incorrect, and present a simple tool that implements this analysis. The goal of this method is to offer a simple extension to the now commonplace decoy database searching, providing additional valuable information.

Modeling of supramolecular structure and dielectric properties of butanols from melting point to supercritical state.

The supramolecular structure and dielectric properties of pure fluid butanols have been modeled in the temperature range from the melting point to the vicinity of the critical point using the quasichemical model of nonideal associated solution (QCNAS). The model of supramolecular structure takes into account both chain-like and cyclic aggregates. Dielectric permittivity and dipole correlation factor of n-, sec-, and tert-butanol are calculated in the temperature intervals 191-553, 166-533, and 298-503 K, respectively, using parameters obtained earlier for ambient conditions. The thermodynamic and structural parameters of supramolecular aggregation are reported. Size and structure distribution functions of aggregates are evaluated in the entire temperature range. Mean degree of aggregation of n-butanol decreases from hundreds at low temperatures to about 1.3 around critical point. The structure of n-butanol and s-butanol is characterized mainly by chain-like aggregation. tert-Butanol's degree of aggregation is about 4 at ambient conditions and falls to 1.4 approaching the critical point. The fractions of molecules in chain-like and cyclic aggregates are comparable for tert-butanol. This conclusion refutes the frequently expressed opinion that tert-butanol consists mostly of cyclic species.

The Paragon Algorithm, a next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra.

The Paragon Algorithm, a novel database search engine for the identification of peptides from tandem mass spectrometry data, is presented. Sequence Temperature Values are computed using a sequence tag algorithm, allowing the degree of implication by an MS/MS spectrum of each region of a database to be determined on a continuum. Counter to conventional approaches, features such as modifications, substitutions, and cleavage events are modeled with probabilities rather than by discrete user-controlled settings to consider or not consider a feature. The use of feature probabilities in conjunction with Sequence Temperature Values allows for a very large increase in the effective search space with only a very small increase in the actual number of hypotheses that must be scored. The algorithm has a new kind of user interface that removes the user expertise requirement, presenting control settings in the language of the laboratory that are translated to optimal algorithmic settings. To validate this new algorithm, a comparison with Mascot is presented for a series of analogous searches to explore the relative impact of increasing search space probed with Mascot by relaxing the tryptic digestion conformance requirements from trypsin to semitrypsin to no enzyme and with the Paragon Algorithm using its Rapid mode and Thorough mode with and without tryptic specificity. Although they performed similarly for small search space, dramatic differences were observed in large search space. With the Paragon Algorithm, hundreds of biological and artifact modifications, all possible substitutions, and all levels of conformance to the expected digestion pattern can be searched in a single search step, yet the typical cost in search time is only 2-5 times that of conventional small search space. Despite this large increase in effective search space, there is no drastic loss of discrimination that typically accompanies the exploration of large search space.

Error-tolerant EST database searches by tandem mass spectrometry and multiTag software.

The MultiTag method (Sunyaev et al., Anal. Chem. 2003 15, 1307-1315) employs multiple error-tolerant searches with peptide sequence tags (Mann and Wilm, Anal. Chem. 1994, 66, 4390-4399) for the identification of proteins from organisms with unsequenced genomes. Here we demonstrate that the error-tolerant capabilities of MultiTag increased the number of peptide alignments and improved the confidence of identifications in an EST database. The MultiTag outperformed conventional database searching software that only utilizes stringent matching of tandem mass spectra to nucleotide sequences of ESTs.

Discovering known and unanticipated protein modifications using MS/MS database searching.

We present an MS/MS database search algorithm with the following novel features: (1) a novel protein database structure containing extensive preindexing and (2) zone modification searching, which enables the rapid discovery of protein modifications of known (i.e., user-specified) and unanticipated delta masses. All of these features are implemented in Interrogator, the search engine that runs behind the Pro ID, Pro ICAT, and Pro QUANT software products. Speed benchmarks demonstrate that our modification-tolerant database search algorithm is 100-fold faster than traditional database search algorithms when used for comprehensive searches for a broad variety of modification species. The ability to rapidly search for a large variety of known as well as unanticipated modifications allows a significantly greater percentage of MS/MS scans to be identified. We demonstrate this with an example in which, out of a total of 473 identified MS/MS scans, 315 of these scans correspond to unmodified peptides, while 158 scans correspond to a wide variety of modified peptides. In addition, we provide specific examples where the ability to search for unanticipated modifications allows the scientist to discover: unexpected modifications that have biological significance; amino acid mutations; salt-adducted peptides in a sample that has nominally been desalted; peptides arising from nontryptic cleavage in a sample that has nominally been digested using trypsin; other unintended consequences of sample handling procedures.