Mass Spectrometry in Proteomics

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.

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!