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.

Use of PASEF for Accelerated Protein Sequence Confirmation and De Novo Sequencing with High Data Quality.

Biopharmaceutical sequences can be well confirmed by multiple protease digests-e.g., trypsin, elastase, and chymotrypsin-followed by LC-MS/MS data analysis. High quality data can be used for de novo sequencing as well. PASEF (Parallel Accumulation and Serial Fragmentation) on the timsTOF instrument has been used to accelerate proteome and protein sequence studies and increase sequence coverage concomitantly.Here we describe the protein chemical and LC-MS methods in detail to generate high quality samples for sequence characterization from only 3 digests. We applied PASEF to generate exhaustive protein sequence coverage maps by combination of results from the three enzyme digests using a short LC gradient. The data quality obtained was high and adequate for determining antibody sequences de novo.Nivolumab and dulaglutide were digested by 3 enzymes individually. For nivolumab, 94/94/90% sequence coverage and 86/84/85% fragment coverage were obtained from the individual digest analysis with trypsin/chymotrypsin/elastase, respectively. For dulaglutide, 96/100/90% sequence coverage and 92/90/83% fragment coverage were obtained. The merged peptide map from the 3 digests for nivolumab resulted in ∼550 peptides; enough to safely confirm the full sequences and to determine the nivolumab sequence de novo.

The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle-down characterization of antibodies.

Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate sequence coverage of the hypervariable regions remains one of the toughest identification challenges by either bottom-up or top-down workflows. Methods that efficiently generate mid-size Ab fragments would further facilitate top-down MS and decrease data complexity. Here, we explore the proteases Cathepsins L and D for forming protein fragments from three IgG1s, one IgG2, and one bispecific, knob-and-hole IgG1. We demonstrate that high-resolution native MS provides a sensitive method for the detection of clipping sites. Both Cathepsins produced multiple, albeit specific cleavages. The Abs were cleaved immediately after the CDR3 region, yielding ~ 12 kDa fragments, that is, ideal sequencing-sized. Cathepsin D, but not Cathepsin L, also cleaved directly below the Ab hinge, releasing the F(ab')2. When constrained by the different disulfide bonds found in the IgG2 subtype or by the tertiary structure of the hole-containing bispecific IgG1, the hinge region digest product was not produced. The Cathepsin L and Cathepsin D clipping motifs were related to sequences of neutral amino acids and the tertiary structure of the Ab. A single pot (L + D) digestion protocol was optimized to achieve 100% efficiency. Nine protein fragments, corresponding to the VL, VH, CL, CH1, CH2, CH3, CL + CH1, and F(ab')2, constituted ~ 70% of the summed intensities of all deconvolved proteolytic products. Cleavage sites were confirmed by the Edman degradation and validated with top-down sequencing. The described work offers a complementary method for middle-down analysis that may be applied to top-down Ab sequencing. ENZYMES: Cathepsin L-EC 3.4.22.15, Cathepsin D-EC 3.4.23.5.

Peak Filtering, Peak Annotation, and Wildcard Search for Glycoproteomics.

Glycopeptides in peptide or digested protein samples pose a number of analytical and bioinformatics challenges beyond those posed by unmodified peptides or peptides with smaller posttranslational modifications. Exact structural elucidation of glycans is generally beyond the capability of a single mass spectrometry experiment, so a reasonable level of identification for tandem mass spectrometry, taken by several glycopeptide software tools, is that of peptide sequence and glycan composition, meaning the number of monosaccharides of each distinct mass, e.g., HexNAc(2)Hex(5) rather than man5. Even at this level, however, glycopeptide analysis poses challenges: finding glycopeptide spectra when they are a tiny fraction of the total spectra; assigning spectra with unanticipated glycans, not in the initial glycan database; and finding, scoring, and labeling diagnostic peaks in tandem mass spectra. Here, we discuss recent improvements to Byonic, a glycoproteomics search program, that address these three issues. Byonic now supports filtering spectra by m/z peaks, so that the user can limit attention to spectra with diagnostic peaks, e.g., at least two out of three of 204.087 for HexNAc, 274.092 for NeuAc (with water loss), and 366.139 for HexNAc-Hex, all within a set mass tolerance, e.g., ± 0.01 Da. Also, new is glycan "wildcard" search, which allows an unspecified mass within a user-set mass range to be applied to N- or O-linked glycans and enables assignment of spectra with unanticipated glycans. Finally, the next release of Byonic supports user-specified peak annotations from user-defined posttranslational modifications. We demonstrate the utility of these new software features by finding previously unrecognized glycopeptides in publicly available data, including glycosylated neuropeptides from rat brain.

Parsimonious Charge Deconvolution for Native Mass Spectrometry.

Charge deconvolution infers the mass from mass over charge (m/z) measurements in electrospray ionization mass spectra. When applied over a wide input m/z or broad target mass range, charge-deconvolution algorithms can produce artifacts, such as false masses at one-half or one-third of the correct mass. Indeed, a maximum entropy term in the objective function of MaxEnt, the most commonly used charge deconvolution algorithm, favors a deconvolved spectrum with many peaks over one with fewer peaks. Here we describe a new "parsimonious" charge deconvolution algorithm that produces fewer artifacts. The algorithm is especially well-suited to high-resolution native mass spectrometry of intact glycoproteins and protein complexes. Deconvolution of native mass spectra poses special challenges due to salt and small molecule adducts, multimers, wide mass ranges, and fewer and lower charge states. We demonstrate the performance of the new deconvolution algorithm on a range of samples. On the heavily glycosylated plasma properdin glycoprotein, the new algorithm could deconvolve monomer and dimer simultaneously and, when focused on the m/z range of the monomer, gave accurate and interpretable masses for glycoforms that had previously been analyzed manually using m/z peaks rather than deconvolved masses. On therapeutic antibodies, the new algorithm facilitated the analysis of extensions, truncations, and Fab glycosylation. The algorithm facilitates the use of native mass spectrometry for the qualitative and quantitative analysis of protein and protein assemblies.

Automated Antibody De Novo Sequencing and Its Utility in Biopharmaceutical Discovery.

Applications of antibody de novo sequencing in the biopharmaceutical industry range from the discovery of new antibody drug candidates to identifying reagents for research and determining the primary structure of innovator products for biosimilar development. When murine, phage display, or patient-derived monoclonal antibodies against a target of interest are available, but the cDNA or the original cell line is not, de novo protein sequencing is required to humanize and recombinantly express these antibodies, followed by in vitro and in vivo testing for functional validation. Availability of fully automated software tools for monoclonal antibody de novo sequencing enables efficient and routine analysis. Here, we present a novel method to automatically de novo sequence antibodies using mass spectrometry and the Supernovo software. The robustness of the algorithm is demonstrated through a series of stress tests. Graphical Abstract ᅟ.

mzML--a community standard for mass spectrometry data.

Mass spectrometry is a fundamental tool for discovery and analysis in the life sciences. With the rapid advances in mass spectrometry technology and methods, it has become imperative to provide a standard output format for mass spectrometry data that will facilitate data sharing and analysis. Initially, the efforts to develop a standard format for mass spectrometry data resulted in multiple formats, each designed with a different underlying philosophy. To resolve the issues associated with having multiple formats, vendors, researchers, and software developers convened under the banner of the HUPO PSI to develop a single standard. The new data format incorporated many of the desirable technical attributes from the previous data formats, while adding a number of improvements, including features such as a controlled vocabulary with validation tools to ensure consistent usage of the format, improved support for selected reaction monitoring data, and immediately available implementations to facilitate rapid adoption by the community. The resulting standard data format, mzML, is a well tested open-source format for mass spectrometer output files that can be readily utilized by the community and easily adapted for incremental advances in mass spectrometry technology.

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.

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.

Efficacy of measuring visual performance of LASIK patients under photopic and mesopic conditions.

INTRODUCTION: The aim of the study was to compare the visual performance of LASIK eyes measured using high-contrast logMAR letter charts under bright (photopic) and dim (mesopic) conditions. MATERIALS AND METHODS: A total of 46 subjects (35 +/- 8 years of age) undergoing LASIK procedures were recruited for the study. The best spectacle-corrected visual acuity (BSCVA) of each subject was measured using the high-contrast ETDRS logMAR chart under photopic and mesopic conditions at 3 visits: preoperative (Pre), 1 month postoperative (Post1) and 3 months postoperative (Post3). The differences in logMAR scores for the right eyes only were analysed for the Pre-Post1 (n = 46), Pre-Post3 (n = 18) and Post1-Post3 (n = 16) comparisons. RESULTS: The logMAR scores of subjects were worse at the 1-month postoperative visit than preoperatively, and improvement in visual performance was seen at the 3-month postoperative visit. These changes in visual performance became more evident under mesopic conditions. The means and standard errors of the differences in logMAR scores for the Pre-Post3 (0.097 +/- 0.020) were slightly larger than those of the Pre-Post1 (-0.067 +/- 0.019) and Post1-Post3 (0.031 +/- 0.012) comparisons. Under mesopic conditions, the visual performance of the subjects was statistically significant for the 3 comparisons, but not under photopic conditions. CONCLUSION: High-contrast logMAR chart performed under mesopic conditions has the potential to replace visual acuity measured under photopic conditions in providing more reliable representation of postoperative visual outcomes of LASIK eyes. Eye doctors should consider performing this vision test routinely to gauge the success of LASIK surgery.

Accommodative stimulus response curve of emmetropes and myopes.

INTRODUCTION: Myopes are suspected to be poorer at responding to accommodative stimuli than emmetropes, and this may worsen the degree of their myopia. The study aims to compare the abilities of young adult emmetropes and myopes in responding to accommodative stimuli, as indicated by their Accommodation Stimulus Response Curves (ASRCs) in a predominantly Chinese population. MATERIALS AND METHODS: Seventeen emmetropes and 33 myopes aged between 16 and 23 years (mean, 18.6 +/- 1.2) were recruited, of whom 11 were progressing and 22 were non-progressing myopes. The ASRC gradients of subjects were measured using the methods of decreasing distance series (DDS), positive (PLS) and negative lens series (NLS). RESULTS: The ASRC is method dependent. The gradients of the curves are significantly different among 3 methods of measurement using single-factor ANOVA (F3.057 = 44.815, P <0.01). The slopes of the accommodative errors of all subjects were steeper using the NLS method, and the lags of accommodation increased with elevated demands. No significant differences in ASRC gradients were found between emmetropes, non-progressing myopes and progressing myopes for the range of accommodative demands for each method. Progressing myopes showed the highest error towards the higher demand compared with the emmetropes and non-progressing myopes. CONCLUSION: Accommodative responses of myopes were more sluggish though there were no statistical differences in ASRC gradients between emmetropes and myopes. It is not certain if the poorer accommodative responses were a cause, or a consequence, of myopia.

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.

Visual experience during cataract surgery: a nation-wide survey on the knowledge of optometry students.

PURPOSE: Many patients experience a variety of visual sensations during cataract surgery under local anaesthesia. Up to 16.2% of patients are frightened by their intraoperative visual experience. This study aimed to determine the knowledge and beliefs of optometry students on this subject. METHODS: A nationwide survey using a standardised, self-administered questionnaire was conducted on all optometry students in Singapore. RESULTS: For cataract surgery under regional anaesthesia, 38.9% of the respondents believed that patients might experience no light perception while 68.5% felt that patients were likely to be able to perceive light. Overall, 70.4% felt that patients might be frightened by their visual experiences and 88.0% believed that preoperative counselling of patients would serve to reduce the fear experienced during the surgery. For cataract surgery under topical anaesthesia, 37.0% of the respondents believed that patients would perceive no light and 74.1% believed that patients would retain light perception. Overall, 67.6% believed that patients may be frightened and 86.1% felt that preoperative counselling would help. CONCLUSION: Many optometry students correctly believed that patients might experience a variety of visual sensations during cataract surgery under local anaesthesia. The majority were also aware that patients might be frightened by this and felt that preoperative counselling would be helpful.

Repeatability of IOLMaster biometry in children.

AIMS: This study compares the repeatability of IOLMaster (Zeiss, Oberkochen, Germany) axial dimension measurements and conventional ultrasonography in children. METHODS: A series of IOLMaster (partial coherence interferometry, optical pachometry) and Echoscan (US 800, Nidek, Tokyo, Japan) (ultrasound) measurements were taken on 179 Chinese children (mean age, 10.6 +/- 0.8 years) taking part in a longitudinal study of myopia development, and the measurements were repeated on 37 of these subjects. RESULTS: IOLMaster axial length measurements showed better repeatability (95% limits of agreement for repeatability, -0.047 to 0.038 mm) than Echoscan axial length measurements (95% limits of agreement for repeatability, -0.85 to 0.67 mm). IOLMaster anterior chamber depth measurements also showed better repeatability (95% limits of agreement for repeatability, -0.053 to 0.073 mm) than Echoscan anterior chamber depth measurements (95% limits of agreement for repeatability, -0.57 to 0.49 mm). IOLMaster measurements were, on average, slightly larger than Echoscan measurements for axial length (by 0.14 mm) and anterior chamber depth (0.09 mm). CONCLUSION: Partial coherence interferometry techniques, such as that used by IOLMaster, should be considered as the standard technique for axial length measurement in children because they are noninvasive, highly precise, and easy to use.