High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry. 2021

Kaiwen Yu, and Zhen Wang, and Zhiping Wu, and Haiyan Tan, and Ashutosh Mishra, and Junmin Peng
Departments of Structural Biology and Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Mass spectrometry (MS)-based proteomic profiling of whole proteome and protein posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome with high throughput and deep coverage. The reproducibility of quantification benefits not only from the fascinating developments in high-performance liquid chromatography (LC) and high-resolution MS with enhanced scan rates but also from the invention of multiplexed isotopic labeling strategies, such as the tandem mass tags (TMT). In this chapter, we introduce a 16-plex TMT-LC/LC-MS/MS protocol for proteomic profiling of biological and clinical samples. The protocol includes protein extraction, enzymatic digestion, PTM peptide enrichment, TMT labeling, and two-dimensional reverse-phase liquid chromatography fractionation coupled with tandem mass spectrometry (MS/MS) analysis, followed by computational data processing. In general, more than 10,000 proteins and tens of thousands of PTM sites (e.g., phosphorylation and ubiquitination) can be confidently quantified. This protocol provides a general protein measurement tool, enabling the dissection of protein dysregulation in any biological samples and human diseases.

UI MeSH Term Description Entries
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D011499 Protein Processing, Post-Translational Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility. Amino Acid Modification, Post-Translational,Post-Translational Modification,Post-Translational Protein Modification,Posttranslational Modification,Protein Modification, Post-Translational,Amino Acid Modification, Posttranslational,Post-Translational Amino Acid Modification,Post-Translational Modifications,Post-Translational Protein Processing,Posttranslational Amino Acid Modification,Posttranslational Modifications,Posttranslational Protein Processing,Protein Processing, Post Translational,Protein Processing, Posttranslational,Amino Acid Modification, Post Translational,Modification, Post-Translational,Modification, Post-Translational Protein,Modification, Posttranslational,Modifications, Post-Translational,Modifications, Post-Translational Protein,Modifications, Posttranslational,Post Translational Amino Acid Modification,Post Translational Modification,Post Translational Modifications,Post Translational Protein Modification,Post Translational Protein Processing,Post-Translational Protein Modifications,Processing, Post-Translational Protein,Processing, Posttranslational Protein,Protein Modification, Post Translational,Protein Modifications, Post-Translational
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D012107 Research Design A plan for collecting and utilizing data so that desired information can be obtained with sufficient precision or so that an hypothesis can be tested properly. Experimental Design,Data Adjustment,Data Reporting,Design, Experimental,Designs, Experimental,Error Sources,Experimental Designs,Matched Groups,Methodology, Research,Problem Formulation,Research Methodology,Research Proposal,Research Strategy,Research Technics,Research Techniques,Scoring Methods,Adjustment, Data,Adjustments, Data,Data Adjustments,Design, Research,Designs, Research,Error Source,Formulation, Problem,Formulations, Problem,Group, Matched,Groups, Matched,Matched Group,Method, Scoring,Methods, Scoring,Problem Formulations,Proposal, Research,Proposals, Research,Reporting, Data,Research Designs,Research Proposals,Research Strategies,Research Technic,Research Technique,Scoring Method,Source, Error,Sources, Error,Strategies, Research,Strategy, Research,Technic, Research,Technics, Research,Technique, Research,Techniques, Research
D005625 Frontal Lobe The part of the cerebral hemisphere anterior to the central sulcus, and anterior and superior to the lateral sulcus. Brodmann Area 8,Brodmann's Area 8,Frontal Cortex,Frontal Eye Fields,Lobus Frontalis,Supplementary Eye Field,Area 8, Brodmann,Area 8, Brodmann's,Brodmanns Area 8,Cortex, Frontal,Eye Field, Frontal,Eye Field, Supplementary,Eye Fields, Frontal,Frontal Cortices,Frontal Eye Field,Frontal Lobes,Lobe, Frontal,Supplementary Eye Fields
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000544 Alzheimer Disease A degenerative disease of the BRAIN characterized by the insidious onset of DEMENTIA. Impairment of MEMORY, judgment, attention span, and problem solving skills are followed by severe APRAXIAS and a global loss of cognitive abilities. The condition primarily occurs after age 60, and is marked pathologically by severe cortical atrophy and the triad of SENILE PLAQUES; NEUROFIBRILLARY TANGLES; and NEUROPIL THREADS. (From Adams et al., Principles of Neurology, 6th ed, pp1049-57) Acute Confusional Senile Dementia,Alzheimer's Diseases,Dementia, Alzheimer Type,Dementia, Senile,Presenile Alzheimer Dementia,Senile Dementia, Alzheimer Type,Alzheimer Dementia,Alzheimer Disease, Early Onset,Alzheimer Disease, Late Onset,Alzheimer Sclerosis,Alzheimer Syndrome,Alzheimer Type Senile Dementia,Alzheimer's Disease,Alzheimer's Disease, Focal Onset,Alzheimer-Type Dementia (ATD),Dementia, Presenile,Dementia, Primary Senile Degenerative,Early Onset Alzheimer Disease,Familial Alzheimer Disease (FAD),Focal Onset Alzheimer's Disease,Late Onset Alzheimer Disease,Primary Senile Degenerative Dementia,Senile Dementia, Acute Confusional,Alzheimer Dementias,Alzheimer Disease, Familial (FAD),Alzheimer Diseases,Alzheimer Type Dementia,Alzheimer Type Dementia (ATD),Alzheimers Diseases,Dementia, Alzheimer,Dementia, Alzheimer-Type (ATD),Familial Alzheimer Diseases (FAD),Presenile Dementia,Sclerosis, Alzheimer,Senile Dementia
D053719 Tandem Mass Spectrometry A mass spectrometry technique using two (MS/MS) or more mass analyzers. With two in tandem, the precursor ions are mass-selected by a first mass analyzer, and focused into a collision region where they are then fragmented into product ions which are then characterized by a second mass analyzer. A variety of techniques are used to separate the compounds, ionize them, and introduce them to the first mass analyzer. For example, for in GC-MS/MS, GAS CHROMATOGRAPHY-MASS SPECTROMETRY is involved in separating relatively small compounds by GAS CHROMATOGRAPHY prior to injecting them into an ionization chamber for the mass selection. Mass Spectrometry-Mass Spectrometry,Mass Spectrometry Mass Spectrometry,Mass Spectrometry, Tandem
D054875 Ubiquitination The act of ligating UBIQUITINS to PROTEINS to form ubiquitin-protein ligase complexes to label proteins for transport to the PROTEASOME ENDOPEPTIDASE COMPLEX where proteolysis occurs. Ubiquitylation
D056148 Chromatography, Reverse-Phase A chromatography technique in which the stationary phase is composed of a non-polar substance with a polar mobile phase, in contrast to normal-phase chromatography in which the stationary phase is a polar substance with a non-polar mobile phase. Chromatography, Reversed-Phase Liquid,Reversed-Phase Chromatography,Reversed-Phase Liquid Chromatography,Reverse-Phase Chromatography,Reverse-Phase Liquid Chromatography,Chromatography, Reverse Phase,Chromatography, Reversed-Phase,Reverse Phase Chromatography,Reversed Phase Chromatography

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