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Related Concept Videos

Mass Analyzers: Common Types01:19

Mass Analyzers: Common Types

The quadrupole mass analyzer consists of four cylindrical metal rods arranged in a diamond carrying a DC voltage and a radio-frequency AC voltage. The motion of ions through the quadrupole depends on the field strength, causing only ions of a certain m/z to resonate successfully and strike the detector at a given field strength. Though the transmission rate for these analyzers is high, the exact elemental composition of the sample is not determined because of low resolution; however, they are...
Tandem Mass Spectrometry01:21

Tandem Mass Spectrometry

Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
Mass Analyzers: Overview01:13

Mass Analyzers: Overview

The mass analyzer is a crucial component of the mass spectrometer. In the ionization chamber, the vaporized sample is bombarded with a high-energy electron beam to generate a radical cation and further fragment into neutral molecules, radicals, and cations. A series of negatively charged accelerator plates accelerate the cations into the mass analyzer. The mass analyzer separates ions according to their mass-to-charge (m/z) ratios and then directs them to the detector. The common types of mass...
Mass Spectrometry: Complex Analysis01:21

Mass Spectrometry: Complex Analysis

Mass spectrometry is an important technique for the identification of pure compounds. However, it has some limitations for the analysis of complex mixtures, often due to excessive fragmentation making the spectrum too complicated to decipher. Mass spectrometry can be combined with suitable separation methods in sequence, forming hyphenated methods, which are useful in the analysis of complex mixtures.
GC–MS is a powerful hyphenated method commonly used in forensics and environmental...
High-Resolution Mass Spectrometry (HRMS)01:15

High-Resolution Mass Spectrometry (HRMS)

The resolution of a mass spectrometer depends on the efficiency of separating ions with different ion masses. The mass of an atom is approximated to the sum of the masses of protons and neutrons inside, considering the masses of protons and neutrons as equal. However, the masses of the proton (1.6726 × 10−24 g) and neutron (1.6749 × 10−24 g) are not truly equal. There is a minor error in the expression of atomic masses relative to the simplest atom of hydrogen. For example, the mass of helium...
Peptide Identification Using Tandem Mass Spectrometry01:33

Peptide Identification Using Tandem Mass Spectrometry

Tandem mass spectrometry, also known as MS/MS or MS2, is an analytical technique that employs two mass analyzers. Essentially it is a series of mass spectrometers that helps isolate a particular biomolecule and then helps study its chemical properties.
This technique helps gather information regarding the protein from which the peptide was obtained and to study the peptides’ amino acid sequence. Identifying peptides from a complex mixture is an important component of the growing field of...

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A Strategy for Sensitive, Large Scale Quantitative Metabolomics
14:18

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Published on: May 27, 2014

Robust and sensitive iTRAQ quantification on an LTQ Orbitrap mass spectrometer.

Marcus Bantscheff1, Markus Boesche, Dirk Eberhard

  • 1Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. marcus.bantscheff@cellzome.com

Molecular & Cellular Proteomics : MCP
|May 31, 2008
PubMed
Summary
This summary is machine-generated.

Optimizing Pulsed Q Dissociation (PQD) on ion trap mass spectrometers enables sensitive peptide quantification. This technique significantly improves protein identification and outperforms other methods for isobaric tag-based proteomics.

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Area of Science:

  • Proteomics
  • Mass Spectrometry
  • Analytical Chemistry

Background:

  • Isobaric stable isotope tagging reagents (e.g., TMT, iTRAQ) allow multiplexed peptide quantification.
  • Ion trap mass spectrometers traditionally have poor low mass fragment recovery, limiting the use of these reagents.
  • Pulsed Q Dissociation (PQD) was developed to address this limitation but faced challenges with fragmentation efficiency.

Purpose of the Study:

  • To demonstrate the practical utility of Pulsed Q Dissociation (PQD) for peptide quantification on ion trap mass spectrometers.
  • To optimize PQD instrument parameters for enhanced low m/z fragment ion intensity.
  • To compare PQD performance against other dissociation techniques and mass spectrometer platforms.

Main Methods:

  • Optimization of key instrument parameters for PQD, including collision energy, activation Q, delay time, ion isolation width, microscans, and trapped ion numbers.
  • Side-by-side comparison of PQD on an LTQ Orbitrap with Collision-Induced Dissociation (CID) on a Q-Tof Ultima using complex protein digests.
  • Comparison of PQD with Higher-Energy Collisional Dissociation (HCD) on an LTQ Orbitrap.
  • Application of iTRAQ quantification using PQD to study drug-target interactions (imatinib in K-562 cells).

Main Results:

  • Optimized PQD parameters enabled accurate peptide quantification at the 100 amol level.
  • PQD on an LTQ Orbitrap quantified twice as many proteins compared to CID on a Q-Tof Ultima, with similar quantification precision (10-15%).
  • PQD on an LTQ Orbitrap demonstrated a lower limit of quantification than HCD on the same instrument.
  • Successful quantitative measurement of kinase interaction profiles using iTRAQ and PQD.

Conclusions:

  • Careful optimization of PQD parameters makes it a practical and highly effective technique for isobaric tag-based peptide quantification on ion trap mass spectrometers.
  • PQD offers superior performance in terms of protein quantification depth and lower limit of quantification compared to traditional CID and HCD in specific configurations.
  • PQD on the LTQ Orbitrap platform has significant potential for quantitative proteomics applications, including drug-target interaction studies.