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.