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Mass Spectrometry of Peptides and Proteins

Elements, Isotopes, and Masses

  • Focus on elements crucial for peptide and protein mass spectrometry: Carbon, Nitrogen, Oxygen, Sulfur, and Hydrogen.
  • Elements form molecules as elemental building blocks.
  • Isotopes: Variations of elements with different numbers of neutrons.
    • Hydrogen isotopes:
      • ^{1}H_{1} (protium): Single proton in the nucleus.
      • ^{2}H_{1} (deuterium): Proton and neutron in the nucleus.
      • ^{3}H_{1} (tritium): Proton and two neutrons in the nucleus.
  • Isotope information:
    • NIST (National Institute of Standards and Technology) provides data on isotopes.
  • Isotope Table Columns:
    • Isotope identification.
    • Relative atomic mass.
    • Isotopic composition (fractional abundance).
  • Isotope Masses:
    • ^{1}H_{1} mass: 1.007825032 Da
    • ^{2}H_{1} mass: 2.014101778 Da
    • ^{3}H_{1} mass: approximately 3 Da
    • ^{12}C mass: exactly 12 Da (basis for atomic mass units).
  • Carbon Isotopes:
    • Carbon-12: Exactly 12 atomic mass units.
    • Carbon-13: Stable isotope.
    • Carbon-14: Radioactive isotope, useful for radiocarbon dating (half-life ~5900 years).
  • Nitrogen Isotopes:
    • Nitrogen-14: Stable isotope.
    • Nitrogen-15: Stable isotope.
  • Oxygen Isotopes:
    • Oxygen-16: Stable isotope.
    • Oxygen-17: Stable isotope.
    • Oxygen-18: Stable isotope.
  • Numbers on the Periodic Table:
    • Weighted averages of isotopic masses based on fractional abundance.
    • Calculated by multiplying the mass of each isotope by its fractional abundance and summing the results.
    • Formula: \text{Average Atomic Mass} = \sum (\% \text{ Abundance} \times \text{Isotopic Mass})
    • Example: Carbon average atomic mass calculation.
  • No single atom of carbon has a mass of 12.0107 Da; atoms are either ^{12}C or ^{13}C.
  • Isotopic composition varies depending on the source of the element.
    • Different carbon sources (atmospheric CO_{2}, plants, animals, fossil fuels) have varying ratios of ^{13}C to ^{12}C.
    • Isotopic ratios provide information about the origin and history of a sample (biogeochemistry).
    • Archaeological applications: Isotope ratios in bones indicate geographical origin based on diet.
    • Coffee bean authentication: Isotopic composition reflects the soil and environmental conditions of the growing region.

Biological Mass Spectrometry

  • Central dogma: DNA → RNA → Proteins.
  • Proteins undergo post-translational modifications (PTMs) affecting function.
  • Mass spectrometry applications:
    • Proteins (Proteomics).
    • DNA (Genomics).
    • Carbohydrates (Glycomics).
    • Lipids (Lipidomics).
    • Metabolites (Metabolomics).

Traditional Molecular Weight Determination

  • Gel Electrophoresis (SDS-PAGE):
    • Proteins migrate through a gel under an electric field based on electrophoretic mobility.
    • Sodium Dodecyl Sulfate (SDS) interacts with positive charges on amino acids.
    • High molecular weight proteins move less; low molecular weight proteins move more.
    • Bands visualized using dyes or stains (e.g., silver staining, Coomassie blue staining).
  • Electrophoretic mobility affected by functional group changes (e.g., phosphorylation) on the protein.
  • SDS-PAGE measurements not always accurate due to charge-based separation.
  • Well-behaved proteins show a linear relationship between mobility and molecular weight.
    • Example of molecular weight vs mobility plot with a straight line.
  • Two-Dimensional Gel Electrophoresis (2D gels):
    • Separates proteins based on isoelectric point (pI) and molecular weight.
    • First dimension: Isoelectric focusing using a pH gradient.
      • Proteins migrate to their isoelectric point where they have no net charge.
    • Second dimension: SDS-PAGE.
  • 2D Gel Applications:
    • Comparing protein expression between samples (e.g., control vs. heat-shocked cells).
    • Identifying proteins with changes in concentration or presence.
    • Spots cut out, extracted, and analyzed by mass spectrometry.
  • Limitations of 2D Gels:
    • Limited sensitivity; staining may not detect low-concentration proteins.
    • Reproducibility issues and labor-intensive.
  • Two-Dimensional Chromatography (2D-HPLC):
    • More reproducible and automatable than 2D gels.
    • Involves two chromatographic separations in orthogonal dimensions.

Mass Spectrometry Approaches

  • Peptide Mass Fingerprinting (PMF):
    • Digest protein with trypsin (or other protease).
    • Trypsin cuts at lysine or arginine residues.
    • Measure masses of tryptic fragments using mass spectrometry.
    • Search a database for proteins matching the observed fragment masses.
    • Bioinformatics tools are used to analyze the data.
  • Peptide Ladder Sequencing:
    • Remove one amino acid at a time from a peptide.
    • Create a ladder of masses corresponding to different length chains.
    • Determine the sequence by measuring the mass differences between ladder steps.
    • Methods for generating the ladder:
      • Enzymatic digestion.
      • Edman sequencing (chemical method).
      • Tandem mass spectrometry (MS/MS).
    • Sequence Tags: Short amino acid sequences used to identify proteins in databases.
    • Sequence Coverage: Percentage of amino acids identified in a protein.

Molecules

  • Amino Acid Example: Phenylalanine
    • Molecular structure vs. residue within a peptide chain.
    • Residue mass is less than the molecular mass due to water loss during peptide bond formation.
    • Mass difference = 18 Da (mass of water).
    • Phenylalanine residue mass: 147.0684 Da.
  • Amino Acid List:
    • One-letter codes, three-letter codes, names, elemental compositions, and masses.
  • Leucine and Isoleucine:
    • Same elemental composition and mass, but different structures.
    • Enzymes can clip chains when leucine is present but not isoleucine.

Proteases

  • Trypsin: Cleaves after lysine or arginine residues.
  • Other proteases with different cleavage specificities.
  • Carboxypeptidases: Remove amino acids from the C-terminal end.
  • Aminopeptidases: Remove amino acids from the N-terminal end.
  • Dipeptidases: Remove dipeptides from a particular end.
  • Enzyme Cocktails: Mixtures of different enzymes for diverse cleavage.
  • Buffer Considerations:
    • Use mass spec friendly buffers.
    • Avoid phosphate buffers, which can contaminate the mass spectrometer.
    • Use high enzyme to substrate ratios for shorter reaction times.

Post Translational Modifications (PTMs)

  • Common PTMs: Phosphorylation, glycosylation, methylation, acetylation.
  • PTMs result in mass changes detectable by mass spectrometry.
  • Used in cell signaling pathways.
  • Resources for PTM information:
    • Brian Chait (Rockefeller University) list of PTMs with masses and sample handling artifacts.
    • Methionine oxidation: Common artifact yielding mass differences of 16 or 32 Da.

Mass Spectrometer Block Diagram

  • Sample Introduction Device (GC, LC, CE, robotic workstation) → Ion Source → Ion Separation Device → Detector → Data System.

Ionization Methods

  • Soft vs. Hard Ionization:
    • Soft ionization: Gentle, produces intact molecular ions with little fragmentation.
    • Hard ionization: Energetic, induces fragmentation.
  • Gas Phase Samples:
    • Electron Ionization (EI): Hard ionization, requires gas phase samples.
    • Chemical Ionization (CI): Soft ionization, requires gas phase samples.
    • Laser Ionization: Requires gas phase samples.
  • Liquid State Samples:
    • Thermospray: Developed in the 1970s for liquid samples.
    • Atmospheric Pressure Chemical Ionization (APCI): Developed in the 1970s for liquid samples.
    • Electrospray Ionization (ESI): Developed in the 1980s for liquid samples.
  • Solid Phase Sampling Methods:
    • Particle Bombardment (Fast Atom Bombardment, Secondary Ion Mass Spectrometry).
    • Laser Desorption.
    • Matrix Assisted Laser Desorption Ionization (MALDI): Developed in the 1980s.

MALDI

  • Analyte coated neat on the probe tip (with nothing else present, it's by itself).
  • UV or IR lasers release neutrals and ions from the surface.
  • Franz Hillenkamp, Michael Karas, around 1985 created MALDI.
  • Analyte dissolved in large excess (1000-10000 fold) of a small organic light absorbing molecule, the matrix.
  • Widely used for peptides, proteins, DNA, carbohydrates, and synthetic polymers.
  • Molecular Weight information comes off intact with little or no fragmentation of adduct formation.
  • MALDI Matrices:
    • Synapinic acid, CHCA, DHB (dihydroxybenzoic acid), Haba, IAA, Dithranol.
    • Aromatic rings for UV absorption (chromophores).
    • Not able to list all properties.
    • Some matrices are better for certain compounds (e.g., CHCA/DHB for peptides, Haba for DNA, Dithranol for hydrophobic polymers).
  • MALDI Process: Solution Phase Sample Preparation
    1. Dissolve solid analyte in a solvent.
    2. Dissolve solid matrix in a solvent.
    3. Mix the solutions to obtain a homogeneous sample solution.
    4. Remove the solvent to create a homogeneous solid solution.
      • Biggest problems in preparing MALDI Sample.
      • Dry drop technique.
      • Recrystallization using impurities inside crystal.
    5. Fire laser.
    6. Ions desorb from the surface; matrix absorbs laser light and is crystalline solids in expansion creates a supersonic expansion for cooling.
    7. Analyze ionization to occur, needs reagent and preference
    8. Time of flight is what's usually used, not required

ElectroSpray Ionization

  • Often paired with high-performance liquid chromatography (HPLC).
  • Effluent from the HPLC flows through a metal needle held at high voltage.
  • Taylor Cone: Liquid forms a cone shape at the needle tip.
  • Tiny droplets (picoliters) are emitted from the tip.
  • Droplets contain the analyte and acid (formic acid, acetic acid).
  • Positively charged droplets are attracted to the negative pole.
  • Droplets evaporate as they move towards the mass spectrometer inlet.
  • Ionization Models:
    • Charge Residue Model: Solvent evaporates completely, leaving charged analyte ions.
    • Ion Evaporation Model: Ions evaporate directly from the droplet surface.
    • Both operate depending on experimental parameters.
  • Types of electrospray sources:
    • pure electrospray.
    • coaxial tubes.
    • nebulizing gas.
    • ultrasonic transducer
  • Solvents have lawyers.
  • MS is about testing samples to see how they perform
  • Metal capillary tube is connected outside to atmosphere pressure and connected inside to vacuum atmosphere.
  • Multiple Charging:
    • Myoglobin example: Multiple peaks due to different numbers of protons attached to the protein.
    • Different charge states: M + nH+ / n, where n is the number of protons.
    • Integer number of protons only.
    • Integer number with software can be calculated.
  • Femtomoles consumed, 20 * 10 to the minus 15 moles.
  • People have gotten to 10 to the minus 18.
  • Samples work between pico 10 to minus 12, nanomole and you can work harder for atomole

Ion Separation Methods

  • Magnetic sector mass spectrometers (1900's):
  • Quadrupoles (1950's -> Triple Quad and Quadrupole Ion Traps).
  • Fourier Transform mass spectrometers (1970's).
  • Time of Flight mass spectrometer (1950's) electrical engineering developing dept at UPenn.
    • Good sensitivity and Unlimited mass rage is theoretical with practical detriments.
    • Delayed extraction gives results.

Time of Flight Mass Spectrometer

  • Advantages over other methods:
    • High sensitivity.
    • Unlimited mass range.
    • Complete mass spectrum is acquired for each ionization pulse.
    • Excellent mass resolution with delayed extraction.
  • Linear Time-of-Flight
    • Metal plate with sample fired with a laser has positive and negative ions released.
    • Metal mesh grid lets ions out with electric field.
    • Kinetic energy = 1/2mv^2: Different mass -> different v for same energy.
    • Lighter ions have higher velocities and travel more quickly.
  • Graham Cook's Diagram
    • y axis voltage across different ion optics
    • x axis for positions: use analogy and then run ion through analogy
    • Ions are balls rolling down hill when running different configurations
  • Wiley and McLaren (1955)
  • Time of Flight
    • two step source increased speed and focused ions at detector for increased ability in space traveling.
  • Reflectron time of flight
    • Ion beam bounces back with voltages in source similar to first model
    • Development by Marmarin, but not shown until 1970's West because soviet union was hard to combine knowledge and trade with.
    • Delayed Extraction = increased experiment and performance.

Tandem Mass Spectrometry

  • Single stage separates then mass analyzer ion, simple single.
  • Tandem (MS/MS) fragments before second analyzer
  • Methods for: collision, photons, surfaces, transfer -> release -> cause, etc.