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.

scholarly journals Matrix-matched calibration curves for assessing analytical figures of merit in quantitative proteomics

2019 ◽  
Author(s):  
Lindsay K Pino ◽  
Han-Yin Yang ◽  
William Stafford Noble ◽  
Brian C Searle ◽  
Andrew N Hoofnagle ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sample Preparation ◽  
Quantitative Proteomics ◽  
Protein Abundance ◽  
Complex Samples ◽  
Figures Of Merit ◽  
Data Independent Acquisition ◽  
Proteomics Experiment ◽  
Series Of Experiments ◽  
Analytical Figures Of Merit

AbstractMass spectrometry is a powerful tool for quantifying protein abundance in complex samples. Advances in sample preparation and the development of data independent acquisition (DIA) mass spectrometry approaches have increased the number of peptides and proteins measured per sample. Here we present a series of experiments demonstrating how to assess whether a peptide measurement is quantitative by mass spectrometry. Our results demonstrate that increasing the number of detected peptides in a proteomics experiment does not necessarily result in increased numbers of peptides that can be measured quantitatively.

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 Noninvasive Analysis Using Data-Independent Acquisition Mass Spectrometry: New Epidermal Proteins That Reveal Sex Differences in the Aging Process

2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Shirui Chen ◽  
Hui Zhang ◽  
Mengting Liu ◽  
Yaochi Wang ◽  
Cong Xin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Sex Differences ◽  
Age Groups ◽  
Protein Composition ◽  
Protein Detection ◽  
High Sensitivity ◽  
Skin Aging ◽  
Protein Biomarkers ◽  
Data Independent Acquisition ◽  
Noninvasive Analysis

The development of mass spectrometry has provided a method with extremely high sensitivity and selectivity that can be used to identify protein biomarkers. Epidermal proteins, lipids, and cornified envelopes are involved in the formation of the skin epidermal barrier. The epidermal protein composition changes with age. Therefore, quantitative proteomic changes may be indicative of skin aging. We sought to utilize data-independent acquisition mass spectrometry for noninvasive analysis of epidermal proteins in healthy Chinese individuals of different age groups and sexes. In our study, we completed high-throughput protein detection, analyzed protein differences with MaxQuant software, and performed statistical analyses of the proteome. We obtained interesting findings regarding ceruloplasmin (CP), which exhibited significant differences and is involved in ferroptosis, a signaling pathway significantly associated with aging. There were also several proteins that differed between sexes in the younger group, but the sex differences disappeared with aging. These proteins, which were associated with both aging processes and sex differences, are involved in signaling pathways such as apoptosis, oxidative stress, and genomic stability and can serve as candidate biomarkers for sex differences during aging. Our approach for noninvasive detection of epidermal proteins and its application to accurately quantify protein expression can provide ideas for future epidermal proteomics studies.

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