Mass Spectrometry in Proteomics

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Questions and Answers

What is the primary focus of proteomics in mass spectrometry?

Analyzing heavy particles under vacuum

Characterizing individual isotopes of elements

Studying the structure of atom nuclei

Measuring changes in protein expression (correct)

Which mass spectrometry method is commonly used for quantitative proteomics?

TOF (correct)

Electrophoresis

Centrifugation

Chromatography

What does the mass to charge ratio (m/z) indicate in mass spectrometry?

The speed of ion movement in a vacuum

The size of the molecule only

The relationship between mass and charge of an ion (correct)

The total number of protons in a molecule

What type of samples can mass spectrometry analyze with high sensitivity?

Trace amounts of proteins Signup and view all the answers

What differentiates isotopes of an element in mass spectrometry?

Their number of neutrons Signup and view all the answers

What mechanism primarily causes droplets to evaporate and fragment in electrospray ionization (ESI)?

Vacuum and electrostatic repulsion Signup and view all the answers

What is the dominant ion mode produced by electrospray ionization (ESI)?

Positive-ion mode Signup and view all the answers

Which factor is primarily responsible for the extent of multiple charging of proteins in ESI mass spectrometry?

Protein geometry in solution Signup and view all the answers

What does the presence of multiple peaks in an ESI mass spectrum of a protein indicate?

Different charge states of the same molecule Signup and view all the answers

What role does HPLC play in the electrospray ionization process?

It serves as a separation step before ionization Signup and view all the answers

Electrospray ionization (ESI) is known for producing which type of molecules?

Intact ionized macromolecules Signup and view all the answers

What is a significant challenge when analyzing proteins with mass spectrometry?

Proteins do not easily vaporize and easily fragment. Signup and view all the answers

Which technique is specifically mentioned as a solution for vaporizing proteins intact in mass spectrometry?

Matrix Assisted Laser Desorption Ionization (MALDI) Signup and view all the answers

What is the role of electrospray ionization (ESI) in mass spectrometry?

To convert the analyte to a gas phase and ionize it. Signup and view all the answers

How does the nominal mass of benzene compare to the monoisotopic mass?

The nominal mass is an integer close to the monoisotopic mass. Signup and view all the answers

What characteristic difference in isotopic peaks is observed in amino acids?

Isotopic peaks are separated by one molecular weight unit. Signup and view all the answers

What is one of the primary reasons proteins are challenging targets for mass spectrometry?

Proteins require a higher energy level to ionize than small molecules. Signup and view all the answers

In ms/ms what is the purpose of the collision cell?

To fragment the ions for further analysis. Signup and view all the answers

What is the typical pressure range of the environment in which ions are analyzed in mass spectrometry?

10-3 to 10-8 mbar Signup and view all the answers

What is one of the primary applications of mass spectrometry in proteomics?

Measuring changes in protein expression Signup and view all the answers

Which mass spectrometry method primarily analyzes small biomolecules and intact proteins through laser-induced desorption?

Matrix-assisted laser desorption/ionization (MALDI) Signup and view all the answers

What is the main principle by which a quadrupole mass spectrometer separates ions?

By oscillating electric and magnetic fields Signup and view all the answers

How does electrospray ionization (ESI) typically create ions from a liquid sample?

By applying a high voltage to cause the formation of droplets that evaporate Signup and view all the answers

In mass spectrometry, what does the acronym TOF stand for, and what is its significance?

Time of flight; it measures the time ions take to travel a set distance Signup and view all the answers

What determines the extent of multiple charging in proteins during electrospray ionization?

The geometric configuration of the protein in solution Signup and view all the answers

Which of the following best describes the ESI mass spectrum of a denatured protein?

Multiple peaks reflecting various charge states of the same protein Signup and view all the answers

In the context of proteomics, what is the primary purpose of HPLC when coupled with ESI?

To purify samples prior to ionization Signup and view all the answers

What type of ions are primarily generated in the positive-ion mode of electrospray ionization?

Positively charged analytes Signup and view all the answers

Which characteristic of ESI contributes to the generation of gas-phase ionized macromolecules?

Vacuum and electrostatic repulsion mechanisms Signup and view all the answers

In electrospray ionization (ESI), which aspect of a protein solution can influence the creation of both polycationic and polyanionic species?

The pH of the protein solution Signup and view all the answers

What is a critical factor affecting the vaporization of proteins in mass spectrometry?

Proteins have high molecular weights. Signup and view all the answers

Which ionization method involves the application of a high potential to a dilute solution of analyte?

Electrospray Ionization (ESI) Signup and view all the answers

What is a primary challenge when using mass spectrometry on proteins compared to small molecules?

Proteins can fragment randomly into small pieces. Signup and view all the answers

Which of the following accurately reflects the role of the collision cell in mass spectrometry?

To introduce energy to promote collisions between ions. Signup and view all the answers

What is the purpose of using Matrix-Assisted Laser Desorption Ionization (MALDI) in protein analysis?

To generate intact ions from large biomolecules. Signup and view all the answers

What distinguishes a TOF analyzer from other mass spectrometry systems?

It measures the speed of ions to determine mass. Signup and view all the answers

Which of the following components is not traditionally part of a mass spectrometry system?

Sample library Signup and view all the answers

What key advantage does electrospray ionization (ESI) provide in mass spectrometry?

It allows for the analysis of large biomolecules without fragmentation. Signup and view all the answers

Study Notes

Mass Spectrometry (MS)

MS analyzes the acceleration of charged analytes in a vacuum.
Analysis yields the mass-to-charge ratio (m/z) of the analyte.
MS provides extremely precise results (up to 1 in 10⁶).
MS is extremely sensitive, detecting ions directly.
MS precisely identifies proteins from trace amounts.
It's a powerful technique for protein or proteome identification, modification analysis, and protein dynamics characterization.

Applications of Protein Mass Spectrometry

Proteomics: Identifying unknowns through proteolytic digestion, peptide mass measurement, and comparison to genome databases.
Quantitative Proteomics: Measuring changes in protein expression.
Protein Sequencing: Determining protein sequences.
Splice Variant Characterization: Identifying splice variants.
Post-translational Modification Characterization: Analyzing post-translational modifications.
Structural Biology: Studying protein structure using native MS and H/D exchange techniques.

MS and Isotopes

In standard chemical labs, many molecules are weighed as a group.
MS weighs molecules individually, although peaks arise from aggregating individual molecules.
MS's high resolution separates individual isotopes of analytes.

Atomic Masses

Atoms comprise electrons and a nucleus.
The nucleus (protons and neutrons) contributes most of the atom's mass.
The strong nuclear force overcomes the Coulomb repulsion of protons, holding the nucleus together.
Elements exist as various isotopes with the same number of protons (Z) but different numbers of neutrons (N).
Isotopes are differentiated by A, where A = Z + N.
Examples include ¹²C (A = 12, Z = 6, N = 6) and ¹⁴C (A = 14, Z = 6, N = 8).

Atomic Masses and Nuclear Mass Defect

Atomic mass unit (amu) is 1/12th the mass of a ¹²C atom (1.6606 × 10^-27 kg).
¹²C = 12 (exactly, by definition).
Mass of a proton (p): 1.0073 amu.
Mass of a neutron (n): 1.0087 amu.
The sum of the masses of constituent particles is greater than the mass of an atom.
The missing mass corresponds to the binding energy of the nucleus (E = mc²).

Most Biologically Abundant Elements

Most biologically abundant elements are nearly monoisotopic.
Monoisotopic mass sums exact masses of the most abundant naturally occurring stable isotope of each atom in the molecule.
Examples include H, C, N, O, and P. Sulfur is an exception.

Atomic Masses (Data)

Atomic mass is the average mass weighted by natural abundances of the isotopes of an element.
Examples of atomic masses for elements like boron (B), carbon (C), nitrogen (N), oxygen (O), fluorine (F), aluminum (Al), silicon (Si), phosphorous (P), chlorine (Cl), and others are shown in the data tables.

Molar Masses

Molar mass is calculated by summing the atomic masses of all constituent atoms in a molecule.
In samples with a large number of molecules, each isotope's average abundance is important in each position.
Example: Benzene (C₆H₆), molar mass = 78.11 g/mol.

Exact Monoisotopic Masses

In MS, exact masses of individual molecules are measured.
The weight is the sum of the exact masses of isotopes in the molecule.
Example: Benzene (C₆H₆), if made of ¹²C and ¹H, the monoisotopic mass is 78.0468.
Nominal mass is a convenient representation of the nearest integer mass; example Benzene (C₆H₆)=78 Da

MS and Isotopes (Examples)

Examples of MS spectra (Bradykinin, Melittin, Ubiquitin) demonstrating the presence of isotopic peaks, important in accurate mass determination and identification.

ESI (Electrospray Ionization)

ESI is an alternative ion source.
It uses a dilute analyte solution with electrical conductivity, sprayed from a metal capillary at high potential (kV), resulting in fragmentation of the analyte, and leaving intact gas-phase analyte molecules carrying a large net charge.
This method is routinely coupled to HPLC (High-Pressure Liquid Chromatography) as a separation step.
ESI is important in protein analysis because it allows intact proteins to be analyzed.

Net Charge in ESI

ESI creates gas-phase ionized macromolecules.
Positive ion mode is predominant.
Multiple charging of unfolded polypeptides is limited by the number of available ionizable sites, but the extent of multiple charging is determined by the protein geometry.
Both polycationic and polyanionic species can be produced.

Mass Analyzer: Quadrupole

A quadrupole uses four bar electrodes to create a complex electromagnetic field.
The field varies sinusoidally, influencing ion trajectories based on m/z.
The field can be tuned to allow only specific m/z ions to have stable trajectories.

Time of Flight (TOF) Mass Analyzer

TOF mass analyzers use an electrostatic field to accelerate ions.
Ions travel through a field-free region with constant velocity.
The travel time is diagnostic of the m/z.
A detector measures the time delay to determine m/z.

Reflectrons for improved TOF MS Precision

MALDI-TOF precision is limited by initial random velocity from the laser pulse.
A reflectron, an electrostatic mirror, helps correct this by delaying ions with higher velocities.

MALDI (Matrix-assisted Laser Desorption/Ionization)

A solid matrix (e.g., sinapinic acid) incorporates a sample protein.
A UV laser vaporizes and ionizes the matrix.
The matrix material provides ionization, making it important in protein analysis, usually for intact protein analysis.
This is an alternative method to ESI for analysis of intact proteins.

MALDI TOF (Time-of-Flight)

MALDI TOF is highly sensitive (1 picomoles detection).
Has high resolution (~20,000).
Mass accuracy is excellent (1 per 100,000) for large proteins.

Resolution and Resolving Power in Mass Spectrometry

Resolving power defines the ability to separate peaks based on m/z differences.
A common metric is the full width at half maximum (FWHM) of the peak, used to define resolution.

Collision-Induced Dissociation (CID) in MS/MS

A precise molecular mass reveals elemental composition, not detailed structure.
CID (in MS/MS) uses a collision gas in a cell to fragment the ion of interest (the precursor ion) for further analysis.
The fragment masses correlate with the parent ion's connectivity.

Triple Quadrupole

A triple quadrupole (instrument used in MS/MS) has three independent quadrupoles in a row.
Q1 and Q3 act as mass filters.
Q2 is the collision cell.

Quadrupole Time-of-Flight (TOF) Mass Spectrometers

Instruments that use multiple stages combined. One includes a quadrupole to filter selected mass-to-charge ions followed by a TOF component that determines time-of-flight from different ions with specific m\z to measure mass.

Detector: Electron Multiplier

An electron multiplier amplifies the charge signal associated with ions.
It is analogous to a photomultiplier.
The signal from the detector is highly sensitive, producing an exquisite level of sensitivity, and allows for extremely small amounts of sample to be analyzed.

Determining the MW of Peptides

Peptide mass determination using MS is straightforward compared to proteins.
By determining the charge (z) and m/z, using the equation [M + nH]/z = n, the MW can be calculated.

Proteomics Overview

The number of proteins produced by an organism is vastly more than the number of genes (due to post-translational modifications, and alternative splicing).
Protein concentrations differ significantly (10 orders of magnitude).

Peptide Analysis in Proteins

MS peak identification for proteins is sometimes problematic due to several proteins having similar masses.
Proteins can be digested using trypsin to yield peptides that are more easily analyzed.
The genome needs to be sequenced to confirm the peptide analyses.

Confirming Peptide Sequence

Techniques like LC-MS/MS, used to confirm peptide sequences.
Specific fragmentation and analysis patterns suggest the sequence, including fragment ions.

Locating PTM Sites

Methods of locating post-translational modification sites using MS/MS, specifically by looking for mass differences, used in structural elucidation of proteins.

Isotope Labeling

Isotopic Labeling allows for relative protein quantification.
This technique distinguishes different samples of the same protein using different isotope-labeled amino acids.

Measuring Protein Level Changes

Comparing protein levels between two samples with LC-MS/MS data. This can be done by measuring differences in protein abundance between samples, helping to identify changes in protein levels. The results are usually displayed in a specialized graph called a volcano plot and shows significant changes in protein levels.

Mass Spectrometry (Summary)

MS converts molecules to gas phase ions (high vacuum), separating based on m/z (electromagnetic lenses).
Quantitative analysis (sensitive & specific), detection in complex mixtures.
Structural information (fragmentation) from MS/MS & soft ionization for intact proteins.
Proteomic identification from tryptic peptide masses, quantitative analysis with labeled standards.

Mass Spectrometry Strength & Weakness

Strengths: Extremely high specificity/sensitivity, fast analysis, wide applicable range of analytes (small-large molecules), easily interfaced with separation technologies.
Weaknesses: Expensive, delicate equipment, sophisticated computing needed, analysis only, not preparative, susceptible to interferences, matrix effects.

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Description

This quiz explores the principles and applications of mass spectrometry (MS) in the field of proteomics. You'll learn about the techniques used to identify proteins, analyze post-translational modifications, and characterize splice variants. Test your knowledge on how MS contributes to understanding protein dynamics and structure!