Mass spectrometry-based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Abstract Quantitative mass spectrometry approaches are used for absolute and relative quantification in global proteome studies. To date, relative and absolute quantification techniques are available that differ in quantification accuracy, proteome coverage, complexity and robustness. This review focuses on most common relative or absolute quantification strategies exemplified by three experimental studies. A label-free relative quantification approach was performed for the investigation of the membrane proteome of sensory cilia to the depth of olfactory receptors in Mus musculus. A SILAC-based relative quantification approach was successfully applied for the identification of core components and transient interactors of the peroxisomal importomer in Saccharomyces cerevisiae. Furthermore, AQUA using stable isotopes was exemplified to unraveling the prenylome influenced by novel prenyltransferase inhibitors. Characteristic enrichment and fragmentation strategies for a robust quantification of the prenylome are also summarized.

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Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Analytical Biochemistry ◽

10.1016/j.ab.2010.10.023 ◽

2011 ◽

Vol 409 (2) ◽

pp. 202-212 ◽

Cited By ~ 11

Author(s):

Melkamu Getie-Kebtie ◽

Alexander Lazarev ◽

Maryna Eichelberger ◽

Michail Alterman

Keyword(s):

Mass Spectrometry ◽

Gel Electrophoresis ◽

Relative Quantification ◽

Label Free ◽

One Dimensional

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Statistical Analysis of Spectral Count Data Generated by Label-Free Tandem Mass Spectrometry-Based Proteomics

Neuromethods - Neuroproteomics ◽

10.1007/978-1-61779-111-6_21 ◽

2011 ◽

pp. 297-309

Author(s):

Thang V. Pham ◽

Connie R. Jimenez

Keyword(s):

Mass Spectrometry ◽

Statistical Analysis ◽

Tandem Mass Spectrometry ◽

Count Data ◽

Tandem Mass ◽

Label Free ◽

Spectral Count

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Comparison of LFQ and IPTL for Protein Identification and Relative Quantification

Molecules ◽

10.3390/molecules26175330 ◽

2021 ◽

Vol 26 (17) ◽

pp. 5330

Author(s):

Christina Johannsen ◽

Christian J. Koehler ◽

Bernd Thiede

Keyword(s):

Protein Identification ◽

Isotopic Labeling ◽

Metabolic Labeling ◽

Relative Quantification ◽

Label Free ◽

Label Free Quantification ◽

Stable Isotopic ◽

Quantification Accuracy ◽

The Many ◽

Free Quantification

(1) Background: Mass spectrometry-based quantitative proteome profiling is most commonly performed by label-free quantification (LFQ), stable isotopic labeling with amino acids in cell culture (SILAC), and reporter ion-based isobaric labeling methods (TMT and iTRAQ). Isobaric peptide termini labeling (IPTL) was described as an alternative to these methods and is based on crosswise labeling of both peptide termini and MS2 quantification. High quantification accuracy was assumed for IPTL because multiple quantification points are obtained per identified MS2 spectrum. A direct comparison of IPTL with other quantification methods has not been performed yet because IPTL commonly requires digestion with endoproteinase Lys-C. (2) Methods: To enable tryptic digestion of IPTL samples, a novel labeling for IPTL was developed that combines metabolic labeling (Arg-0/Lys-0 and Arg-d4/Lys-d4, respectively) with crosswise N-terminal dimethylation (d4 and d0, respectively). (3) Results: The comparison of IPTL with LFQ revealed significantly more protein identifications for LFQ above homology ion scores but not above identity ion scores. (4) Conclusions: The quantification accuracy was superior for LFQ despite the many quantification points obtained with IPTL.

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Relative and Absolute Quantitation in Mass Spectrometry–Based Proteomics

Annual Review of Analytical Chemistry ◽

10.1146/annurev-anchem-061516-045357 ◽

2018 ◽

Vol 11 (1) ◽

pp. 49-77 ◽

Cited By ~ 36

Author(s):

J. Astor Ankney ◽

Adil Muneer ◽

Xian Chen

Keyword(s):

Mass Spectrometry ◽

Mass Spectrum ◽

Quantitative Proteomics ◽

Label Free ◽

Absolute Quantitation ◽

Chemical Labeling ◽

Stable Isotope Label ◽

Proteome Coverage ◽

Isotope Label ◽

Quantification Accuracy

Mass spectrometry–based quantitative proteomics is a powerful tool for gaining insights into function and dynamics of biological systems. However, peptides with different sequences have different ionization efficiencies, and their intensities in a mass spectrum are not correlated with their abundances. Therefore, various label-free or stable isotope label–based quantitation methods have emerged to assist mass spectrometry to perform comparative proteomic experiments, thus enabling nonbiased identification of thousands of proteins differentially expressed in healthy versus diseased cells. Here, we discuss the most widely used label-free and metabolic-, enzymatic-, and chemical labeling–based proteomic strategies for relative and absolute quantitation. We summarize the specific strengths and weaknesses of each technique in terms of quantification accuracy, proteome coverage, multiplexing capability, and robustness. Applications of each strategy for solving specific biological complexities are also presented.

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On the beta-binomial model for analysis of spectral count data in label-free tandem mass spectrometry-based proteomics

Bioinformatics ◽

10.1093/bioinformatics/btp677 ◽

2009 ◽

Vol 26 (3) ◽

pp. 363-369 ◽

Cited By ~ 119

Author(s):

Thang V. Pham ◽

Sander R. Piersma ◽

Marc Warmoes ◽

Connie R. Jimenez

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Count Data ◽

Binomial Model ◽

Tandem Mass ◽

Label Free ◽

Spectral Count

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The use of label-free mass spectrometry for relative quantification of sarcoplasmic proteins during the processing of dry-cured ham

Food Chemistry ◽

10.1016/j.foodchem.2015.09.062 ◽

2016 ◽

Vol 196 ◽

pp. 437-444 ◽

Cited By ~ 24

Author(s):

Marta Gallego ◽

Leticia Mora ◽

M. Concepción Aristoy ◽

Fidel Toldrá

Keyword(s):

Mass Spectrometry ◽

Relative Quantification ◽

Label Free ◽

Sarcoplasmic Proteins ◽

Dry Cured Ham

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freeQuant: A Mass Spectrometry Label-Free Quantification Software Tool for Complex Proteome Analysis

The Scientific World JOURNAL ◽

10.1155/2015/137076 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Ning Deng ◽

Zhenye Li ◽

Chao Pan ◽

Huilong Duan

Keyword(s):

Mass Spectrometry ◽

Functional Analysis ◽

Quantitative Analysis ◽

Protein Quantification ◽

Mouse Heart ◽

Label Free ◽

Spectral Count ◽

Label Free Quantification ◽

Mass Spectrometry Technology ◽

Free Quantification

Study of complex proteome brings forward higher request for the quantification method using mass spectrometry technology. In this paper, we present a mass spectrometry label-free quantification tool for complex proteomes, called freeQuant, which integrated quantification with functional analysis effectively. freeQuant consists of two well-integrated modules: label-free quantification and functional analysis with biomedical knowledge. freeQuant supports label-free quantitative analysis which makes full use of tandem mass spectrometry (MS/MS) spectral count, protein sequence length, shared peptides, and ion intensity. It adopts spectral count for quantitative analysis and builds a new method for shared peptides to accurately evaluate abundance of isoforms. For proteins with low abundance, MS/MS total ion count coupled with spectral count is included to ensure accurate protein quantification. Furthermore, freeQuant supports the large-scale functional annotations for complex proteomes. Mitochondrial proteomes from the mouse heart, the mouse liver, and the human heart were used to evaluate the usability and performance of freeQuant. The evaluation showed that the quantitative algorithms implemented in freeQuant can improve accuracy of quantification with better dynamic range.

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