9.4 Mass spectrometry for biomolecules

Mass spectrometry is a powerful tool for analyzing biomolecules. It measures the mass-to-charge ratio of ions, helping scientists determine molecular mass and structure. This technique is crucial for identifying proteins, studying modifications, and examining complexes.

Various ionization methods like electrospray and MALDI make it possible to analyze large molecules. Mass spectrometry's high sensitivity and ability to handle complex mixtures make it invaluable in biophysical research, despite some challenges in sample preparation and quantification.

Mass Spectrometry Principles

Basics of Mass Spectrometry

• Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z) of ions to determine the molecular mass and chemical structure of biomolecules
• The basic components of a mass spectrometer include an ionization source, mass analyzer, detector, and data processing system
• Biomolecules are ionized and converted into gas-phase ions, which are then separated based on their m/z values in the mass analyzer
• The detector records the abundance of ions at each m/z value, generating a mass spectrum that provides information about the molecular mass and relative abundance of the biomolecules

Applications of Mass Spectrometry in Biophysical Research

• MS is widely used in biophysical research for protein identification, characterization of post-translational modifications, analysis of protein complexes, and studying protein-ligand interactions
  • Protein identification involves matching the observed mass spectrum to a database of known protein sequences (UniProt, NCBI)
  • Post-translational modifications (phosphorylation, glycosylation) can be identified by the mass shifts they cause in the spectrum
  • Protein complexes can be analyzed by native MS to determine stoichiometry and binding interactions
  • Protein-ligand interactions can be studied by hydrogen-deuterium exchange MS (HDX-MS) to map binding sites and conformational changes
• MS can also be applied to the analysis of other biomolecules, such as nucleic acids, carbohydrates, and lipids
  • Nucleic acids (DNA, RNA) can be sequenced and characterized by MS, especially when coupled with enzymatic digestion
  • Carbohydrates can be analyzed by MS to determine their composition, sequence, and branching patterns
  • Lipids can be profiled and quantified by MS to study their roles in biological membranes and signaling pathways

Ionization Techniques in Mass Spectrometry

Electrospray Ionization (ESI)

• Electrospray ionization (ESI) is a soft ionization technique that produces multiply charged ions from biomolecules in solution
  • In ESI, the sample solution is passed through a capillary at high voltage, creating a fine spray of charged droplets that evaporate to form gas-phase ions
  • The formation of multiply charged ions (M+nH)^n+ enables the analysis of large biomolecules within the mass range of the instrument
• ESI is suitable for analyzing large, non-volatile biomolecules, such as proteins and nucleic acids, and is compatible with liquid chromatography (LC-MS)
  • LC-MS couples the separation power of liquid chromatography with the sensitivity and specificity of mass spectrometry
  • LC-MS is widely used for proteomics, metabolomics, and drug discovery applications

Matrix-Assisted Laser Desorption/Ionization (MALDI)

• Matrix-assisted laser desorption/ionization (MALDI) is another soft ionization technique that uses a laser to desorb and ionize biomolecules co-crystallized with a matrix
  • The matrix absorbs the laser energy and facilitates the ionization of the biomolecules, typically producing singly charged ions (M+H)^+
  • Common matrices include α-cyano-4-hydroxycinnamic acid (CHCA) for peptides and sinapinic acid (SA) for proteins
• MALDI is often used for analyzing peptides, proteins, and other large biomolecules, and is compatible with time-of-flight (TOF) mass analyzers
  • MALDI-TOF MS is a rapid and sensitive method for peptide mass fingerprinting and protein identification
  • MALDI imaging MS enables the spatial mapping of biomolecules in tissue sections, providing insights into their distribution and localization

Other Ionization Techniques

• Atmospheric pressure chemical ionization (APCI) is an ionization technique that uses gas-phase ion-molecule reactions to ionize small, relatively non-polar molecules
• Fast atom bombardment (FAB) is an older ionization technique that uses a beam of high-energy atoms to desorb and ionize biomolecules from a liquid matrix
• Surface-enhanced laser desorption/ionization (SELDI) is a variant of MALDI that uses functionalized surfaces to selectively capture and analyze specific classes of biomolecules

Mass Spectra Interpretation

Components of a Mass Spectrum

• A mass spectrum is a plot of ion abundance (intensity) versus m/z values, representing the molecular mass and relative abundance of the detected ions
• The base peak is the most abundant ion in the spectrum, and its intensity is set to 100%. The relative intensities of other ions are calculated with respect to the base peak
• The molecular ion peak (M+) corresponds to the intact, ionized biomolecule and provides information about its molecular mass
  • For singly charged ions, the m/z value of the molecular ion peak directly represents the molecular mass
  • For multiply charged ions, the molecular mass can be calculated from the m/z values and charge states of the observed ions
• Fragment ion peaks result from the dissociation of the molecular ion and provide structural information about the biomolecule
  • Fragmentation can occur through various mechanisms, such as collision-induced dissociation (CID) or electron capture dissociation (ECD)
  • The fragmentation pattern depends on the type of biomolecule and the dissociation method used

Identification and Characterization of Biomolecules

• Post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, and acetylation, can be identified by the mass shifts they cause in the spectrum
  • Phosphorylation adds a mass of 79.97 Da per phosphate group
  • Glycosylation can add various masses depending on the type and number of sugar residues
  • Acetylation adds a mass of 42.01 Da per acetyl group
• Tandem mass spectrometry (MS/MS) involves the selection and fragmentation of specific ions, generating a fragmentation pattern that aids in the identification and characterization of biomolecules and their modifications
  • In peptide sequencing, MS/MS spectra are used to determine the amino acid sequence based on the mass differences between fragment ions
  • In glycan analysis, MS/MS spectra provide information about the composition, sequence, and linkage of sugar residues
  • In lipid analysis, MS/MS spectra enable the identification of lipid classes, fatty acyl chains, and head group modifications

Strengths vs Weaknesses of Mass Spectrometry

Strengths of Mass Spectrometry

• Mass spectrometry offers high sensitivity, specificity, and ability to analyze complex mixtures of biomolecules
  • MS can detect and identify biomolecules at femtomole to attomole levels, making it suitable for analyzing low-abundance proteins and other biomolecules
  • The high mass accuracy and resolution of modern mass spectrometers enable the precise determination of molecular mass and the differentiation of closely related species
• MS can be coupled with separation techniques, such as liquid chromatography (LC) and capillary electrophoresis (CE), to analyze complex biological samples
  • LC-MS and CE-MS enable the separation and identification of hundreds to thousands of biomolecules in a single analysis
  • Multidimensional separations, such as 2D-LC-MS or LC-CE-MS, further enhance the resolving power and coverage of the analysis

Weaknesses of Mass Spectrometry

• Mass spectrometry often requires extensive sample preparation and can be affected by matrix effects and ion suppression
  • Sample preparation, such as protein digestion and desalting, can be time-consuming and may introduce artifacts or biases
  • Matrix effects and ion suppression can occur when co-eluting compounds interfere with the ionization of the analytes, leading to reduced sensitivity and accuracy
• MS-based quantification can be challenging, especially for absolute quantification, and often requires the use of internal standards or label-free approaches
  • Stable isotope labeling (SILAC, iTRAQ) enables relative quantification but requires specific sample preparation and data analysis workflows
  • Label-free quantification relies on the comparison of ion intensities or spectral counts but may be affected by variations in ionization efficiency and sample complexity
• Some biomolecules, such as highly hydrophobic or low-abundance proteins, may be difficult to analyze by MS due to their poor ionization efficiency or low concentration in the sample
  • Membrane proteins and other hydrophobic biomolecules may require specialized sample preparation or ionization techniques (detergents, nanodiscs)
  • Low-abundance proteins may necessitate extensive fractionation or enrichment steps to enable their detection and identification by MS