Evaluation of Acquisition Modes for the Quantitative Analysis of Cross-Linked Peptides by Targeted and Untargeted Mass Spectrometry

2020 ◽  
Author(s):  
Hannah Britt ◽  
Tristan Cragnolini ◽  
Suniya Khatun ◽  
Abubakar Hatimy ◽  
Juliette James ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Range ◽  
Temporal Dynamics ◽  
Protein Complexes ◽  
Isotopic Labeling ◽  
Single Experiment ◽  
Data Independent Acquisition ◽  
Quantitative Metrics ◽  
Parallel Reaction Monitoring ◽  
Further Development

<div> <div> <p>Cross-linking mass spectrometry (XL-MS) is a structural biology technique that can provide insights into the structure and interactions of proteins and their complexes, especially those that cannot be easily assessed by other methods. Quantitative XL-MS has the potential to probe the structural and temporal dynamics of protein complexes; however, it requires further development. Until recently, quantitative XL-MS has largely relied upon isotopic labeling and data dependent acquisition (DDA) methods, limiting the number of biological samples that can be studied in a single experiment. Here, the acquisition modes available on an ion mobility (IM) enabled QToF mass spectrometer are evaluated for the quantitation of cross-linked peptides, eliminating the need for isotopic labels and thus expanding the number of comparable studies that can be conducted in parallel. Workflows were optimized using metabolite and peptide standards analyzed in biological matrices, facilitating modelling of the data and addressing linearity issues, which allow for significant increases in dynamic range. Evaluation of the DDA acquisition method commonly used in XL-MS studies indicated consistency issues between technical replicates and reduced performance in quantitative metrics. On the contrary, data independent acquisition (DIA) and parallel reaction monitoring (PRM) modes proved more robust for analyte quantitation. Mobility enabled modes exhibited an improvement in sensitivity due to the added dimension of separation, and a simultaneous reduction in dynamic range, which was largely recovered by correction methods. Hi[3] and probabilistic quantitation methods were successfully applied to the DIA data, determining the molar amounts of cross-linked peptides relative to their linear counterparts.</p></div></div>

Evaluation of Acquisition Modes for the Quantitative Analysis of Cross-Linked Peptides by Targeted and Untargeted Mass Spectrometry

2020 ◽  
Author(s):  
Hannah Britt ◽  
Tristan Cragnolini ◽  
Suniya Khatun ◽  
Abubakar Hatimy ◽  
Juliette James ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Range ◽  
Temporal Dynamics ◽  
Protein Complexes ◽  
Isotopic Labeling ◽  
Single Experiment ◽  
Data Independent Acquisition ◽  
Quantitative Metrics ◽  
Parallel Reaction Monitoring ◽  
Further Development

<div> <div> <p>Cross-linking mass spectrometry (XL-MS) is a structural biology technique that can provide insights into the structure and interactions of proteins and their complexes, especially those that cannot be easily assessed by other methods. Quantitative XL-MS has the potential to probe the structural and temporal dynamics of protein complexes; however, it requires further development. Until recently, quantitative XL-MS has largely relied upon isotopic labeling and data dependent acquisition (DDA) methods, limiting the number of biological samples that can be studied in a single experiment. Here, the acquisition modes available on an ion mobility (IM) enabled QToF mass spectrometer are evaluated for the quantitation of cross-linked peptides, eliminating the need for isotopic labels and thus expanding the number of comparable studies that can be conducted in parallel. Workflows were optimized using metabolite and peptide standards analyzed in biological matrices, facilitating modelling of the data and addressing linearity issues, which allow for significant increases in dynamic range. Evaluation of the DDA acquisition method commonly used in XL-MS studies indicated consistency issues between technical replicates and reduced performance in quantitative metrics. On the contrary, data independent acquisition (DIA) and parallel reaction monitoring (PRM) modes proved more robust for analyte quantitation. Mobility enabled modes exhibited an improvement in sensitivity due to the added dimension of separation, and a simultaneous reduction in dynamic range, which was largely recovered by correction methods. Hi[3] and probabilistic quantitation methods were successfully applied to the DIA data, determining the molar amounts of cross-linked peptides relative to their linear counterparts.</p></div></div>

scholarly journals Proteomic insights into synaptic signaling in the brain: the past, present and future

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yalan Xu ◽  
Xiuyue Song ◽  
Dong Wang ◽  
Yin Wang ◽  
Peifeng Li ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Complexes ◽  
Synaptic Signaling ◽  
Brain Functions ◽  
The Past ◽  
Data Independent Acquisition ◽  
Technological Advances ◽  
Neural Communication ◽  
Chemical Synapses ◽  
The Brain

AbstractChemical synapses in the brain connect neurons to form neural circuits, providing the structural and functional bases for neural communication. Disrupted synaptic signaling is closely related to a variety of neurological and psychiatric disorders. In the past two decades, proteomics has blossomed as a versatile tool in biological and biomedical research, rendering a wealth of information toward decoding the molecular machinery of life. There is enormous interest in employing proteomic approaches for the study of synapses, and substantial progress has been made. Here, we review the findings of proteomic studies of chemical synapses in the brain, with special attention paid to the key players in synaptic signaling, i.e., the synaptic protein complexes and their post-translational modifications. Looking toward the future, we discuss the technological advances in proteomics such as data-independent acquisition mass spectrometry (DIA-MS), cross-linking in combination with mass spectrometry (CXMS), and proximity proteomics, along with their potential to untangle the mystery of how the brain functions at the molecular level. Last but not least, we introduce the newly developed synaptomic methods. These methods and their successful applications marked the beginnings of the synaptomics era.

Variable-Temperature Native Mass Spectrometry for Studies of Protein Folding, Stabilities, Assembly, and Molecular Interactions

2021 ◽  
Vol 51 (1) ◽  
Author(s):  
Arthur Laganowsky ◽  
David E. Clemmer ◽  
David H. Russell
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Range ◽  
Protein Complexes ◽  
Noncovalent Interactions ◽  
Variable Temperature ◽  
Regulatory Protein ◽  
Conformational Dynamics ◽  
Native Mass Spectrometry ◽  
Annual Review ◽  
Publication Date

The structures and conformational dynamics of proteins, protein complexes, and their noncovalent interactions with other molecules are controlled specifically by the Gibbs free energy (entropy and enthalpy) of the system. For some organisms, temperature is highly regulated, but the majority of biophysical studies are carried out at room, nonphysiological temperature. In this review, we describe variable-temperature electrospray ionization (vT-ESI) mass spectrometry (MS)-based studies with unparalleled sensitivity, dynamic range, and selectivity for studies of both cold- and heat-induced chemical processes. Such studies provide direct determinations of stabilities, reactivities, and thermodynamic measurements for native and non-native structures of proteins and protein complexes and for protein–ligand interactions. Highlighted in this review are vT-ESI-MS studies that reveal 40 different conformers of chymotrypsin inhibitor 2, a classic two-state (native → unfolded) unfolder, and thermochemistry for a model membrane protein system binding lipid and its regulatory protein. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals NAguideR: performing and prioritizing missing value imputations for consistent bottom-up proteomic analyses

2020 ◽  
Vol 48 (14) ◽  
pp. e83-e83 ◽  
Author(s):  
Shisheng Wang ◽  
Wenxue Li ◽  
Liqiang Hu ◽  
Jingqiu Cheng ◽  
Hao Yang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Missing Values ◽  
Protein Complexes ◽  
Label Free ◽  
Missing Value ◽  
Imputation Methods ◽  
Data Independent Acquisition ◽  
Low Performance ◽  
User Friendly

Abstract Mass spectrometry (MS)-based quantitative proteomics experiments frequently generate data with missing values, which may profoundly affect downstream analyses. A wide variety of imputation methods have been established to deal with the missing-value issue. To date, however, there is a scarcity of efficient, systematic, and easy-to-handle tools that are tailored for proteomics community. Herein, we developed a user-friendly and powerful stand-alone software, NAguideR, to enable implementation and evaluation of different missing value methods offered by 23 widely used missing-value imputation algorithms. NAguideR further evaluates data imputation results through classic computational criteria and, unprecedentedly, proteomic empirical criteria, such as quantitative consistency between different charge-states of the same peptide, different peptides belonging to the same proteins, and individual proteins participating protein complexes and functional interactions. We applied NAguideR into three label-free proteomic datasets featuring peptide-level, protein-level, and phosphoproteomic variables respectively, all generated by data independent acquisition mass spectrometry (DIA-MS) with substantial biological replicates. The results indicate that NAguideR is able to discriminate the optimal imputation methods that are facilitating DIA-MS experiments over those sub-optimal and low-performance algorithms. NAguideR further provides downloadable tables and figures supporting flexible data analysis and interpretation. NAguideR is freely available at http://www.omicsolution.org/wukong/NAguideR/ and the source code: https://github.com/wangshisheng/NAguideR/.

scholarly journals Insights Into the Polerovirus–Plant Interactome Revealed by Coimmunoprecipitation and Mass Spectrometry

2015 ◽  
Vol 28 (4) ◽  
pp. 467-481 ◽  
Author(s):  
Stacy L. DeBlasio ◽  
Richard Johnson ◽  
Jaclyn Mahoney ◽  
Alexander Karasev ◽  
Stewart M. Gray ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Fixation ◽  
Dynamic Range ◽  
High Resolution Mass Spectrometry ◽  
Protein Complexes ◽  
Interaction Network ◽  
Host Protein ◽  
Host Proteins ◽  
Leafroll Virus ◽  
Readthrough Protein

Identification of host proteins interacting with the aphidborne Potato leafroll virus (PLRV) from the genus Polerovirus, family Luteoviridae, is a critical step toward understanding how PLRV and related viruses infect plants. However, the tight spatial distribution of PLRV to phloem tissues poses challenges. A polyclonal antibody raised against purified PLRV virions was used to coimmunoprecipitate virus-host protein complexes from Nicotiana benthamiana tissue inoculated with an infectious PLRV cDNA clone using Agrobacterium tumefaciens. A. tumefaciens-mediated delivery of PLRV enabled infection and production of assembled, insect-transmissible virus in most leaf cells, overcoming the dynamic range constraint posed by a systemically infected host. Isolated protein complexes were characterized using high-resolution mass spectrometry and consisted of host proteins interacting directly or indirectly with virions, as well as the nonincorporated readthrough protein (RTP) and three phosphorylated positional isomers of the RTP. A bioinformatics analysis using ClueGO and STRING showed that plant proteins in the PLRV protein interaction network regulate key biochemical processes, including carbon fixation, amino acid biosynthesis, ion transport, protein folding, and trafficking.

scholarly journals Data-independent acquisition mass spectrometry enables reproducible characterization of microbiota function

2018 ◽  
Author(s):  
Juhani Aakko ◽  
Sami Pietilä ◽  
Tomi Suomi ◽  
Mehrad Mahmoudian ◽  
Raine Toivonen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dynamic Range ◽  
Functional Characterization ◽  
Research Area ◽  
Microbial Composition ◽  
Complex Samples ◽  
Data Independent Acquisition ◽  
Novel Approach ◽  
Open Source Software Package

AbstractMetaproteomics is an emerging research area which aims to reveal the functionality of microbial communities – unlike the increasingly popular metagenomics providing insights only on the functional potential. So far, the common approach in metaproteomics has been data-dependent acquisition mass spectrometry (DDA). However, DDA is known to have limited reproducibility and dynamic range with samples of complex microbial composition. To overcome these limitations, we introduce here a novel approach utilizing data-independent acquisition (DIA) mass spectrometry, which has not been applied in metaproteomics of complex samples before. For robust analysis of the data, we introduce an open-source software package diatools, which is freely available at Docker Hub and runs on various operating systems. Our highly reproducible results on laboratory-assembled microbial mixtures and human fecal samples support the utility of our approach for functional characterization of complex microbiota. Hence, the approach is expected to dramatically improve our understanding on the role of microbiota in health and disease.

scholarly journals Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment

2015 ◽  
Vol 24 (8) ◽  
pp. 1292-1300 ◽  
Author(s):  
Albert Konijnenberg ◽  
Ludovic Bannwarth ◽  
Duygu Yilmaz ◽  
Armağan Koçer ◽  
Catherine Venien-Bryan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Sequence Information ◽  
Oligomeric State ◽  
Top Down ◽  
Single Experiment ◽  
Intact Membrane ◽  
Membrane Protein Complexes ◽  
Top Down Mass Spectrometry

scholarly journals Development of Targeted Mass Spectrometry-Based Approaches for Quantitation of Proteins Enriched in the Postsynaptic Density (PSD)

Proteomes ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 12 ◽  
Author(s):  
Rashaun Wilson ◽  
Navin Rauniyar ◽  
Fumika Sakaue ◽  
TuKiet Lam ◽  
Kenneth Williams ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Postsynaptic Density ◽  
Protein Composition ◽  
Protein Abundance ◽  
Large Network ◽  
Glutamatergic Synapses ◽  
Data Independent Acquisition ◽  
Parallel Reaction Monitoring ◽  
Biochemical Fractionation ◽  
Internal Peptide

The postsynaptic density (PSD) is a structural, electron-dense region of excitatory glutamatergic synapses, which is involved in a variety of cellular and signaling processes in neurons. The PSD is comprised of a large network of proteins, many of which have been implicated in a wide variety of neuropsychiatric disorders. Biochemical fractionation combined with mass spectrometry analyses have enabled an in-depth understanding of the protein composition of the PSD. However, the PSD composition may change rapidly in response to stimuli, and robust and reproducible methods to thoroughly quantify changes in protein abundance are warranted. Here, we report on the development of two types of targeted mass spectrometry-based assays for quantitation of PSD-enriched proteins. In total, we quantified 50 PSD proteins in a targeted, parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards and identified and quantified over 2100 proteins through a pre-determined spectral library using a data-independent acquisition (DIA) approach in PSD fractions isolated from mouse cortical brain tissue.

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