This work presents a detailed analysis of glycopeptides produced in the tryptic digestion of an IgG1 reference material. Analysis was done by nanospray ESI LC–MS/MS over a wide range of HCD collision ...energies with both conventional 1D separation for various digestion conditions and a 20 fraction 2D-LC study of a single digest. An extended version of NIST-developed software for analysis of “shotgun” proteomics served to identify the glycopeptides from their precursor masses and product ions for peptides with up to three missed cleavages. A peptide with a single missed cleavage, TKPREEQYNSTYR, was dominant and led to the determination of almost all glycans reported in this study. The 2D studies found a total of 247 glycopeptide ions and 60 glycans of different masses, including 30 glycans found in the 1D studies. This significantly larger number of glycans than found in any other glycoanalysis of therapeutic glycoproteins is due to both the improved separation of sialylated versus asialylated species in the first (high-pH) dimension and the ability to inject large amounts of glycosylated peptides in the 2D studies. Systematic variations in retention with glycan size were also noted. Energy-dependent changes in HCD fragmentation confirmed the proposed glycan structures and led to a peak-annotated mass spectral library to aid the analysis of glycopeptides derived from IgG1 drugs.
Rationale
The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different liquid chromatography/mass spectrometry (LC/MS) platforms showed significant differences. ...Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross‐platform chemical signatures.
Methods
Non‐targeted global metabolite profiles of SRM 1950 were obtained on different LC/MS platforms using reversed‐phase chromatography and different chromatographic scales (conventional HPLC, UHPLC and nanoLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in‐house software (NIST pipeline).
Results
Repeatability and intermediate precision showed that the non‐targeted SRM 1950 profiling was highly reproducible when working on the same platform (relative standard deviation (RSD) <2%); however, substantial differences were found in the LC/MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed.
Conlusions
Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross‐platform transferable. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.
The first full-scale software implementation of the dynamic data evaluation concept {ThermoData Engine (TDE)} is described for thermophysical property data. This concept requires the development of ...large electronic databases capable of storing essentially all experimental data known to date with detailed descriptions of relevant metadata and uncertainties. The combination of these electronic databases with expert-system software, designed to automatically generate recommended data based on available experimental data, leads to the ability to produce critically evaluated data dynamically or ‘to order'. Six major design tasks are described with emphasis on the software architecture for automated critical evaluation including dynamic selection and application of prediction methods and enforcement of thermodynamic consistency. The direction of future enhancements is discussed.
Inhibitors of ataxia–telangiectasia mutated (ATM), such as KU‐55933 (Ku), represent a promising class of novel anticancer drugs. In addition, the biguanide derivative phenformin exhibits antitumor ...activity superior to that of the AMPK activator metformin. Herein, we assessed the potential combinatorial therapeutic efficacy of phenformin and Ku when used to inhibit the growth of liver cancer cells, and we assessed the mechanisms underlying such efficacy. The Hep‐G2 and SMMC‐7721 liver cancer cell lines were treated with phenformin and Ku either alone or in combination, after which the impact of these drugs on cellular proliferation was assessed via 3‐(4,5‐dimethylthiazol) 2, 5‐diphenyltetrazolium and colony formation assays, whereas Transwell assays were used to gauge cell migratory activity. The potential synergy between these two drugs was assessed using the CompuSyn software, while flow cytometry was employed to evaluate cellular apoptosis. In addition, western blotting was utilized to measure p‐ATM, p‐AMPK, p‐mTOR, and p‐p70s6k expression, while mitochondrial functionality was monitored via morphological analyses, JC‐1 staining, and measurements of ATP levels. Phenformin and Ku synergistically impacted the proliferation, migration, and apoptotic death of liver cancer cells. Together, these compounds were able to enhance AMPK phosphorylation while inhibiting the phosphorylation of mTOR and p70s6k. These data also revealed that phenformin and Ku induced mitochondrial dysfunction as evidenced by impaired ATP synthesis, mitochondrial membrane potential, and abnormal mitochondrial morphology. These findings suggest that combination treatment with phenformin and Ku may be an effective approach to treating liver cancer via damaging mitochondria within these tumor cells.
Phenformin and ku‐55933 have synergistic effects on inhibition of proliferation, migration, and apoptosis of liver cancer cell lines. These two compounds stimulate p‐AMPK protein and inhibit the expression of p‐mTOR and p‐p70s6k. Furthermore, phenformin and ku‐55933 induced mitochondrial dysfunction as evidenced by decreased ATP synthesis, mitochondrial membrane potential, and disrupted mitochondrial morphology.
We present UniSpec, an attention-driven deep neural network designed to predict comprehensive collision-induced fragmentation spectra, thereby improving peptide identification in shotgun proteomics. ...Utilizing a training data set of 1.8 million unique high-quality tandem mass spectra (MS2) from 0.8 million unique peptide ions, UniSpec learned with a peptide fragmentation dictionary encompassing 7919 fragment peaks. Among these, 5712 are neutral loss peaks, with 2310 corresponding to modification-specific neutral losses. Remarkably, UniSpec can predict 73%–77% of fragment intensities based on our NIST reference library spectra, a significant leap from the 35%–45% coverage of only b and y ions. Comparative studies with Prosit elucidate that while both models are strong at predicting their respective fragment ion series, UniSpec particularly shines in generating more complex MS2 spectra with diverse ion annotations. The integration of UniSpec’s predictions into shotgun proteomics data analysis boosts the identification rate of tryptic peptides by 48% at a 1% false discovery rate (FDR) and 60% at a more confident 0.1% FDR. Using UniSpec’s predicted in-silico spectral library, the search results closely matched those from search engines and experimental spectral libraries used in peptide identification, highlighting its potential as a stand-alone identification tool. The source code and Python scripts are available on GitHub (https://github.com/usnistgov/UniSpec) and Zenodo (https://zenodo.org/records/10452792), and all data sets and analysis results generated in this work were deposited in Zenodo (https://zenodo.org/records/10052268).
e12601
Background: Previous studies have shown that the tumor regression shrinkage pattern (SP) of triple-negative breast cancer (TNBC) during neoadjuvant chemotherapy (NAC) is more likely to present ...with concentric shrinkage (CS), which is associated with a pathological complete response (pCR). Since immunotherapy and chemotherapy have different antitumor mechanisms, it is reasonable to believe that there may be different SPs between neoadjuvant immunochemotherapy (NAIC) and NAC. This NeoMDSS-TN-SP prospective study aimed to investigate whether tumor SPs differed between NAIC and NAC in TNBC, as well as to calculate the correlation between SP and efficacy. Methods: In this prospective, single-center, nonrandomized cohort study, eligible patients with stage II-III TNBC received NAIC or NAC and underwent MRI at baseline, after the first cycle of neoadjuvant therapy, and preoperatively. Based on the MRI after the first cycle, the tumor SPs were grouped into three categories: CS, diffuse decrease, and no change or enlargement. The primary end point was the difference in SP between the two treatment groups. Secondary endpoints included the correlation between SP and treatment efficacy. This study is registered with ClinicalTrials.gov, number NCT04909554. Results: Ninety-nine patients were enrolled from January 2019 to July 2021; 65 received NAC and 34 received NAIC. Baseline characteristics were similar between the two groups. More patients in the NAIC group achieved pCR after surgery than in the NAC group (82.4% vs. 44.6%, p=0.0003). In the NAC group, more patients showed CS (53.8%), and patients with the CS pattern had a significantly higher pCR rate than those with diffuse decrease or no change/enlargement (65.7% vs. 21.7% vs. 14.3%, p=0.0007). In the NAIC group, although a higher proportion of patients showed diffuse decrease (67.6%), all three SP categories had relatively high pCR rates, so pCR was not correlated with SP (85.7% vs. 82.6% vs. 75.0%, p=0.9029). Conclusions: This is the first study to investigate the tumor regression SP of NAIC in TNBC patients. Compared to traditional chemotherapy, NAIC was more prone to diffuse regression, but the relatively high pCR rates had no correlation with the SPs during neoadjuvant therapy. Clinical trial information: NCT04909554 . Table: see text
Surgical phase recognition is a fundamental task in computer-assisted surgery systems. Most existing works are under the supervision of expensive and time-consuming full annotations, which require ...the surgeons to repeat watching videos to find the precise start and end time for a surgical phase. In this paper, we introduce timestamp supervision for surgical phase recognition to train the models with timestamp annotations, where the surgeons are asked to identify only a single timestamp within the temporal boundary of a phase. This annotation can significantly reduce the manual annotation cost compared to the full annotations. To make full use of such timestamp supervisions, we propose a novel method called uncertainty-aware temporal diffusion (UATD) to generate trustworthy pseudo labels for training. Our proposed UATD is motivated by the property of surgical videos, i.e ., the phases are long events consisting of consecutive frames. To be specific, UATD diffuses the single labelled timestamp to its corresponding high confident ( i.e ., low uncertainty) neighbour frames in an iterative way. Our study uncovers unique insights of surgical phase recognition with timestamp supervision: 1) timestamp annotation can reduce 74% annotation time compared with the full annotation, and surgeons tend to annotate those timestamps near the middle of phases; 2) extensive experiments demonstrate that our method can achieve competitive results compared with full supervision methods, while reducing manual annotation costs; 3) less is more in surgical phase recognition, i.e ., less but discriminative pseudo labels outperform full but containing ambiguous frames; 4) the proposed UATD can be used as a plug-and-play method to clean ambiguous labels near boundaries between phases, and improve the performance of the current surgical phase recognition methods. Code and annotations obtained from surgeons are available at https://github.com/xmed-lab/ TimeStamp-Surgical.
This paper reviews practices in the expression of uncertainty in the experimental literature for thermodynamic property measurements with determinations of critical temperature for pure compounds ...used as a case study. The time period considered is from 1940 to the present, with an emphasis on results published since 1990. One hundred ninety-four articles were considered involving hundreds of compounds. The goals of this paper are to show how the nature and extent of estimations of uncertainty have changed in this time period and to document the extent to which the information provided in the articles corresponds to the recommendations of the GUM. (The GUM is the Guide to the Expression of Uncertainty in Measurement published in 1993 by the International Organization for Standardization, ISO. This document describes current recommended practices in this area.) It is shown that although gradual and continuous progress has been made in the reporting of uncertainty information, comprehensive uncertainty analyses remain rare, particularly with regard to the consideration of contributions arising from sample impurities. Problems in the application of experimental property results to technological challenges are discussed within the broad context of inconsistencies in the expression of uncertainty. Widespread acceptance and application of the standard methods and terminologies established within the GUM are encouraged for the benefit of experimentalists, data evaluators, and data users alike.
Metabolomics has a critical need for better tools for mass spectral identification. Common metabolites may be identified by searching libraries of tandem mass spectra, which offers important ...advantages over other approaches to identification. But tandem libraries are not nearly complete enough to represent the full molecular diversity present in complex biological samples. We present a novel hybrid search method that can help identify metabolites not in the library by similarity to compounds that are. We call it “hybrid” searching because it combines conventional, direct peak matching with the logical equivalent of neutral-loss matching. A successful hybrid search requires the library to contain “cognates” of the unknown: similar compounds with a structural difference confined to a single region of the molecule, that does not substantially alter its fragmentation behavior. We demonstrate that the hybrid search is highly likely to find similar compounds under such circumstances.
This set of six volumes provides a systematic and standardized description for 23,033 chemical components isolated from 6734 medicinal plants. It provides a chemical structure with stereo-chemistry ...bonds for each chemical component.
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