Mass Spectrometry Fundamentals

Mass Spectrometry

• Mass spectrometry (MS) analyzes the acceleration of charged analytes within a vacuum electrical field.
• Analysis determines the mass-to-charge ratio (m/z) of the analyte.
• MS results are extremely precise, up to 1 in 10⁶, due to the absence of friction and diffusion in a vacuum.
• MS excels at sensitivity as ions are detected directly.
• It accurately identifies proteins from trace amounts, making it a potent analytical tool.
• MS can be used to identify proteins or proteomics, identify modifications, and characterize protein dynamics.

Applications of Protein Mass Spectrometry

• Proteomics: Identifying unknown proteins through proteolytic digestion, peptide mass measurement, and comparison to genome databases.
• Quantitative Proteomics: Measuring changes in protein expression.
• Protein Sequencing: Determining the amino acid sequence of proteins.
• Characterizing Splice Variants and Post-Translational Modifications: Investigating protein variations and modifications.
• Structural Biology: Studying protein structure using native MS or hydrogen/deuterium exchange.

MS and Isotopes

• In conventional "wet" labs, large numbers of molecules are weighed collectively.
• In contrast, MS weighs individual molecules, even as peaks arise from the contributions of many molecules.
• MS's high resolution allows for the resolution of individual isotopes of analytes.

Atomic Masses

• Atoms consist of electrons and a nucleus.
• The nucleus comprises neutrons and protons, contributing most of the atom's mass.
• Nuclei are bound by the strong nuclear force, overriding the Coulomb repulsion of protons.
• Each element comprises various isotopes, with the same number of protons (Z) but different numbers of neutrons (N).
• Isotopes are distinguished by A (atomic mass), where A = Z + N.
• Examples: 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, equivalent to 1.6606 × 10^-27 kg.
• ¹²C has a mass of 12 by definition.
• Proton mass: 1.0073 amu
• Neutron mass: 1.0087 amu
• The mass of an atom is less than the sum of its constituent particles.
• The missing mass represents the binding energy of the nucleus, as per E = mc².

Most Biologically Abundant Elements

• Monoisotopic mass represents the sum of the exact masses of the most abundant naturally occurring stable isotopes of each atom within a molecule.
• Biological molecules primarily comprise light elements (C, H, O, N, S, P).
• Most light elements are nearly monoisotopic:
  • Hydrogen (¹H): 99.99%
  • Carbon (¹²C): 98.9%
  • Nitrogen (¹⁴N): 99.6%
  • Oxygen (¹⁶O): 99.8%
  • Phosphorus (³¹P): 100%
  • Sulfur: an exception with multiple isotopes (³²S, ³³S, ³⁴S, ³⁶S).

Atomic Masses (Elements)

• Atomic mass is the average mass of an element's isotopes, weighted by their natural abundances.
• Example: Chlorine (Cl) weighted average for ³⁵Cl (75.8% abundance) and ³⁷Cl (24.2% abundance).

Molar Masses

• Molecular mass is calculated by summing the atomic masses of all constituent atoms in a molecule.
• In large samples, each isotope exists at its average abundance.
• Example: Benzene (C₆H₆), molar mass = 78.11.

Exact Monoisotopic Masses

• In MS, molecular masses of individual molecules are measured.
• Molecular weight is the sum of exact masses for all isotopes present.
• Exact monoisotopic mass differs slightly from molar mass for molecules of interest.
• Example: Benzene’s exact monoisotopic mass using ¹²C and ¹H: 78.0468.

MS and Isotopes (Proteins)

• Mass spectra of proteins or peptides show isotopic peaks that differ by 1 Da.
• Multiple mass spectrometry steps are used.
• Sample examples demonstrating this pattern for Bradykinin, Melittin, and Ubiquitin.

ESI (Electrospray Ionization)

• ESI produces gas-phase, intact, ionized macromolecules for analysis.
• Typically produces positively charged analytes.
• The extent of multiple charging stems from the protein's geometry, not solely the ionizable sites.

Ion source: Electrospray ionization

• Analyte is buffered (dilute solution) for conductivity.
• The solution is sprayed from a metal capillary.
• A high potential is applied to the capillary.
• Vacuum and electrostatic repulsion cause repeated fragmentation and evaporation.
• The result of this is naked analyte molecules are produced with a large net charge.
• This is a powerful ionization method routinely used with HPLC for sample separation.

Electrospray Ionization (ESI) Diagram of Function

• Shows the process of protein solution with nebulizing gas, going through; thermal desolvation, collisional desolvation, ion focusing, mass analyzer, and injection to finalize.
• This diagram illustrates protein samples being separated using an HPLC beforehand and then being subject to injection into the mass spectrometer.

Net Charge in ESI

• Multiple charge carriers, including protons (especially after HPLC) and alkali metal cations, like Na⁺ and K⁺ are observed.
• Calculation of m/z values for ions.
• Charging typically results from protonation, simplifying calculations.

Direct Calculation of Charge

• Spacing between isotopes in a protein mass spectrum is typically 1 Da.
• The charge (n) can be calculated using m/z values for consecutive isotopes.
• Mathematically deconvoluted MS spectra provides the molecule's mass.

MALDI (Matrix-Assisted Laser Desorption/Ionization)

• Protein samples dissolved with material matrix are then spotted on a metal plate and allowed to dry.
• Matrix material absorbs UV light (from e.g., a nitrogen laser) which vaporizes the matrix material, carrying the protein into the gas phase and forming positively charged ions.

MALDI ToF (Time of Flight)

• High sensitivity, ~1 picomole is typically sufficient.
• High resolution, 20,000 maximum in most cases.
• Highly accurate mass measurements are possible.
• Ideal for complicated samples.
• A common method to analyze the data from the mass spectrograph.

Resolution and Resolving Power

• Resolving power describes distinctions between ions with slight mass-to-charge (m/z) ratio differences.
• Defined by the ratio of the m/z value to the difference in the m/z.

Mass Analyzer: Quadrupole

• Quadrupoles use four bar electrodes to form a complex electromagnetic field.
• The field's sinusoidal pattern dictates ion trajectories based on m/z.
• Tunable field isolates particular m/z values, excluding others.
• Suitable for a full spectrum recording.

Time of Flight (TOF) Mass Analyzer

• Accelerated ionized particles travel through a drift tube, which is not affected by electric fields.
• Travel time is inversely proportional to the particle's m/z or the mass-to-charge ratio.
• A detector measures the arrival times, converting them to m/z values.
• High voltage electric field is used in the process.

TOF Analysis

• The kinetic energy of the particle can be determined using the potential difference across the electrostatic field.
• Derived from the particle’s m/z which is related to time, length of the tube, and voltage.

Reflectrons to Improve TOF Mass Spectrometry Precision

• Reflectron helps correct inaccuracies resulting from variable initial velocities post-laser pulse.

Practice Question (Solution)

• For a 10+ charged ion with 192kDa mass and a 2m drift tube, an acceleration potential of 10 kV is used.
• Calculated time of traversal for the ion.

The Components of a Mass Spectrometer (MS/MS Experiments)

• Shows the components of a mass spectrograph, including an ionization source, mass analyzer, collision cell, ion detector, and additional mass analyzer.

Collision Induced Dissociation (CID) in MS/MS

• Specific ion selection for fragmentation analysis.
• The selected ion enters a collision cell, which leads to random ion fragmentation by collisions with neutral molecules.
• Collision fragmentation patterns give useful insight to the connectivity of the parent ion, and provides structural information.

Application to Protein Analysis

• The mass spectra of the fragmented molecules, provides insights into the structure and connectivity of the original parent ion molecule.

Triple Quadrupole

• The instrument has three quadrupoles, where Q1 and Q3 act as mass filters, while Q2 functions as a collision cell.
• Q1 selects ions based on m/z for dissociation in Q2, with fragmentation product masses detected by Q3.

Quadrupole Time of Flight (Q-TOF) Mass Spectrometer

• Illustrates the instrument's design, labeling different stages of mass spectrometry.
• Parts (source, quadrupole, trap, and transfer sections) are essential for mass spectrometry to function.

Detector: Electron Multiplier

• Analogous to a photomultiplier, the electron multiplier amplifies charge using sequential dynodes in a vacuum.
• Ejection of secondary electrons cascades through each dynode, leading to a high electron gain (around 10⁸).

Determining the Molecular Weight (MW) of Peptides

• Easier to determine than proteins with smaller molecule sizes and simplified analysis.
• Examples of methods, including calculation of the charge, calculating the molecular weight, and using the formula for m/z.

Locating the Sites of PTMs (Post-translational Modifications)

• Methods used to determine where modifications take place in proteins.
• Analysis involves looking at the characteristic MS patterns to identify these changes.

Isotope Labelling

• Allows for the distinction and quantification of different protein forms with different masses or modifications, even from the same protein.
• Cells are grown in media containing isotopes with heavier masses, then analyzed using mass spectrometry.

Measuring Protein Level Changes

• LC-MS/MS (liquid chromatography-tandem mass spectrometry) is used for determining and measuring protein amount changes.
• These methods, such as "volcano plots", can also quantify, in some cases, thousands of proteins simultaneously.

Structural Characterization – HDX-MS (Hydrogen/Deuterium Exchange)

• Analyzes the different forms of proteins to understand their structures.
• This process is also known as hydrogen/deuterium exchange.
• Helps determine and locate structural elements within a protein, in a structured or unstructured fashion.

Progress to Date (HDX-MS)

• Shows how the process of hydrogen/deuterium exchange is utilized to measure structural changes to proteins during hydrogen/deuterium exchange mass spectrometry over time.

Mass Spectrometry Summary

• Molecules are turned into gas-phase ions under vacuum and separated using electromagnetic lenses.
• Quantitative analyte detection is highly sensitive in complex mixtures.
• Precise measurement of elemental composition is possible through isotopic peaks.
• Structural information can be gained through fragmentation patterns.

Mass Spectrometry Strengths/Weaknesses

• Strengths: Extremely high specificity and sensitivity, applicability to diverse analytes including small molecules and macromolecules, seamless integration with other separation techniques.
• Weaknesses: High cost, need for specialized laboratories, expertise, sensitivity to interferences, which makes the output quantitative and preparative difficult.

This quiz explores key concepts of mass spectrometry, including its primary measurements, sensitivity, and the significance of isotopes and proteins. Test your knowledge on the methods used in protein mass spectrometry and the role of various components within a mass spectrometer.