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Concept: Tandem mass tags

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Isobaric labeling strategies (e.g. iTRAQ or TMT) are commonly applied in tandem mass spectrometric (MS/MS) level quantitative proteomics. However, we frequently observed missing isotope reporter ion signals in a large-scale liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric (LC/MALDI-TOF/TOF) quantitative proteomics experiment. To understand this issue, we systematically investigated the processing of MS/MS spectra into peak lists prior to peptide identification and quantification.

Concepts: Scientific method, Mass spectrometry, Research, Proteomics, Tandem mass spectrometry, Tandem mass tags, Quantitative proteomics, SILAC

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Isobaric labeling strategies, such as isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tags (TMT), have promised to dramatically increase the power of quantitative proteomics. However, when applied to complex mixtures, both the accuracy and precision are undermined by interfering peptide ions that coisolate and cofragment with the target peptide. Additional gas-phase isolation steps, such as proton-transfer ion-ion reactions (PTR) or higher-order MS3 scans, can almost completely eliminate this problem. Unfortunately, these methods come at the expense of decreased acquisition speed and sensitivity. Here we present a method that allows accurate quantification of TMT-labeled peptides at the MS2 level without additional ion purification. Quantification is based on the fragment ion cluster that carries most of the TMT mass balance. In contrast to the use of low m/z reporter ions, the localization of these complement TMT (TMT©) ions in the spectrum is precursor-specific; coeluting peptides do not generally affect the measurement of the TMT© ion cluster of interest. Unlike the PTR or MS3 strategies, this method can be implemented on a wide range of high-resolution mass spectrometers like the quadrupole Orbitrap instruments (QExactive). A current limitation of the method is that the efficiency of TMT© ion formation is affected by both peptide sequence and peptide ion charge state; we discuss potential routes to overcome this problem. Finally, we show that the complement reporter ion approach allows parallelization of multiplexed quantification and therefore holds the potential to multiply the number of distinct peptides that can be quantified in a given time frame.

Concepts: Protein, Mass spectrometry, Peptide, Ion, Accuracy and precision, Ion source, Fourier transform ion cyclotron resonance, Tandem mass tags

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Over the past decade, chemical labeling with isobaric tandem mass tags, such as isobaric tags for relative and absolute quantification reagents (iTRAQ) and tandem mass tag (TMT) reagents, has been employed in a wide range of different clinically orientated serum and plasma proteomics studies. In this review the scope of these works is presented with attention to the areas of research, methods employed and performance limitations. These applications have covered a wide range of diseases, disorders and infections, and have implemented a variety of different preparative and mass spectrometric approaches. In contrast to earlier works, which struggled to quantify more than a few hundred proteins, increasingly these studies have provided deeper insight into the plasma proteome extending the numbers of quantified proteins to over a thousand.

Concepts: Proteins, Protein, Mass spectrometry, Proteomics, Proteome, Tandem mass tags

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In this chapter we describe the workflow we use for labeled quantitative proteomics analysis using tandem mass tags (TMT) starting with the sample preparation and ending with the multivariate analysis of the resulting data. We detail the step-by-step process from sample processing, labeling, fractionation, and data processing using Proteome Discoverer through to data analysis and interpretation in the context of a multi-run experiment. The final analysis and data interpretation rely on an R package we call TMTPrepPro, which are deployed on a local GenePattern server, and used for generating various outputs which are also outlined herein.

Concepts: Scientific method, Data, Data analysis, Qualitative research, Test method, Proteomics, Data mining, Tandem mass tags

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Quantitative proteomics using isobaric reagents tandem mass tags (TMT) or isobaric tags for relative and absolute quantitation (iTRAQ) provide a convenient approach to compare changes in protein abundances across multiple samples. However, the analysis of complex protein digests by isobaric labeling can be undermined by the relative large proportion of co-selected peptide ions that lead to distorted reporter ion ratios and affect the accuracy and precision of quantitative measurements. Here, we investigated the use of high-field asymmetric waveform ion mobility spectrometry (FAIMS) in proteomic experiments to reduce sample complexity and improve protein quantification using TMT isobaric labeling. LC-FAIMS-MS/MS analyses of human and yeast protein digests led to significant reduction of interfering ions which increased the number of quantifiable peptides by up to 68 % while significantly improving the accuracy of abundance measurements compared to conventional LC-MS/MS. The improvement of quantitative measurements using FAIMS is further demonstrated for the temporal profiling of protein abundance of HEK293 cells following heat shock treatment.

Concepts: Protein, Improve, Sample size, Mass spectrometry, Hydrogen, Measurement, Proteomics, Tandem mass tags

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Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics.

Concepts: Cancer, Oncology, Medical terms, Proteomics, Marker, Tandem mass tags

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The use of tandem mass tags (TMT) as an isobaric labeling strategy is a powerful method for quantitative proteomics, yet its accuracy has traditionally suffered from interference. This interference can be largely overcome by selecting MS(2) fragment precursor ions for high-energy collision induced dissociation (HCD)-MS(3) analysis in an Orbitrap scan. While this approach minimizes the interference effect, sensitivity suffers due to the high AGC targets and long acquisition times associated with MS(3) Orbitrap detection. We investigated whether acquiring the MS(3) scan in a linear ion trap with its lower AGC target would increase overall quantification levels with a minimal effect on precision and accuracy. Trypsin-digested proteins from Saccharomyces cerevisiae were tagged with 6-plex TMT reagents. The sample was subjected to replicate analyses using either the Orbitrap or the linear ion trap for the HCD-MS(3) scan. HCD-MS(3) detection in the linear ion trap vs. Orbitrap increased protein identification by 66% with minor loss in precision and accuracy. Thus, the use of a linear ion trap - HCD MS(3) scan during a 6-plex TMT experiment can improve overall identification levels while maintaining the power of multiplexed quantitative analysis.

Concepts: Scientific method, Mass spectrometry, Saccharomyces cerevisiae, Saccharomyces pastorianus, Accuracy and precision, Saccharomyces, Fourier transform ion cyclotron resonance, Tandem mass tags

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This pilot study aimed to identify candidate proteins for future study that are differentially expressed in vestibular schwannoma (VS) cerebrospinal fluid (CSF) and to compare such proteins with those previously identified in perilymph and specimen secretions. CSF was collected intraoperatively prior to removal of untreated sporadic VS (3 translabyrinthine, 3 middle cranial fossa approaches) and compared with reference CSF samples. After proteolytic digestion and iTRAQ labeling, tandem mass spectrometry with ProteinPilot was used to identify candidate proteins. Of the 237 proteins detected, 13 were dysregulated in ≥3 of the 6 VS patients versus controls, and 13 were dysregulated (12 up, 1 down) in samples from patients with class D versus class B hearing. Four perilymph proteins of interest were dysregulated in ≥1 VS CSF samples. Thus, 26 candidate VS CSF biomarkers were identified that should be considered in future VS biomarker and tumor pathophysiology investigations.

Concepts: Protein, Metabolism, Mass spectrometry, Proteomics, Biomarker, Cerebrospinal fluid, Tandem mass spectrometry, Tandem mass tags

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The mzQuantML data standard was designed to capture the output of quantitative software in proteomics, to support submissions to public repositories, development of visualisation software and pipeline/modular approaches. The standard is designed around a common core that can be extended to support particular types of technique through the release of semantic rules that are checked by validation software. The first release of mzQuantML supported four quantitative proteomics techniques via four sets of semantic rules: i) intensity-based (MS(1) ) label free, ii) MS(1) label-based (such as SILAC or N(15) ), iii) MS(2) tag-based (iTRAQ or tandem mass tags), and iv) spectral counting. We present an update to mzQuantML for supporting SRM techniques. The update includes representing the quantitative measurements, and associated meta-data, for SRM transitions, the mechanism for inferring peptide-level or protein-level quantitative values, and support for both label-based or label-free SRM protocols, through the creation of semantic rules and controlled vocabulary terms. We have updated the specification document for mzQuantML (version 1.0.1) and the mzQuantML validator to ensure that consistent files are produced by different exporters. We also report the capabilities for production of mzQuantML files from popular SRM software packages, such as Skyline and Anubis. This article is protected by copyright. All rights reserved.

Concepts: Controlled vocabulary, All rights reserved, Support, Copyright, Tandem mass tags, Quantitative proteomics, SILAC, ITRAQ

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Isobaric tagging reagents, such as tandem mass tags (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ), are high-throughput methods that allow the analysis of multiple samples simultaneously, which reduces instrument time and error. Accuracy and precision of isobaric tags are limited, however, in tandem mass spectrometry (MS/MS) acquisition due to co-isolation and co-fragmentation of neighboring peptide peaks in precursor scans. Here we present a MS(3) method using pulsed-Q dissociation (PQD) in ion trap and Orbitrap instrumentation as a means to improve ratio distortion and maintain high numbers of identified and quantified proteins.

Concepts: Mass spectrometry, Accuracy and precision, Tandem mass spectrometry, Fourier transform ion cyclotron resonance, Top-down proteomics, Tandem mass tags, Quantitative proteomics, ITRAQ