What is Protein Sequencing?
Proteins, as the fundamental building blocks of life, are involved in virtually every cellular process. To understand
their roles and functions, it is imperative to determine their sequences accurately. Mass spectrometry has heralded
a revolution in the field of proteomics. It enables researchers to delve deep into the proteomic landscape by
offering a comprehensive view of the proteins present in a sample. Mass spectrometry's precision in measuring the
mass-to-charge ratio of ions is unparalleled. This accuracy is harnessed to uniquely identify proteins based on
their mass spectra, even distinguishing closely related protein isoforms and variants.
Mass Spectrometry Platform in Protein Sequencing
Mass Spectrometer Types:
MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry): MALDI-TOF is commonly
used for analyzing intact proteins and peptides. It uses a laser to ionize and desorb molecules from a solid matrix.
The ions are then accelerated in a time-of-flight analyzer based on their mass-to-charge ratio (m/z).
ESI-MS (Electrospray Ionization Mass Spectrometry): ESI-MS is often used in combination with liquid chromatography
(LC-MS/MS) for peptide analysis. It generates ions by spraying the analyte solution through an electric field,
leading to the formation of charged droplets and subsequent ionization.
Orbitrap MS: Orbitrap mass spectrometers offer high-resolution and accurate mass measurements, making them suitable
for precise protein and peptide identification. They use an electrostatic trapping and detection technique.
FT-ICR MS (Fourier Transform Ion Cyclotron Resonance Mass Spectrometry): FT-ICR instruments provide high mass
accuracy and resolution. They trap ions in a magnetic field and measure their oscillation frequencies to determine
mass.
Ionization Sources:
MALDI (Matrix-Assisted Laser Desorption/Ionization): MALDI sources use a laser to vaporize the matrix and create
ions. It is suitable for analyzing larger biomolecules, including proteins.
ESI (Electrospray Ionization): ESI sources generate ions by applying a high voltage to a liquid sample. It is
commonly used for peptide analysis in LC-MS/MS workflows.
Mass Analyzers:
Time-of-Flight (TOF): TOF analyzers measure the time it takes for ions to travel a known distance. They are used in
MALDI-TOF instruments for rapid analysis.
Quadrupole: Quadrupole analyzers use radiofrequency and DC voltages to selectively pass ions of specific m/z values.
They are often used for precursor ion selection in tandem MS.
Ion Trap: Ion traps trap and store ions, allowing for multiple stages of fragmentation and analysis. They are common
in ion trap mass spectrometers.
Orbitrap: Orbitrap analyzers use the motion of ions in an electrostatic field to measure their mass with high
accuracy and resolution.
FT-ICR (Fourier Transform Ion Cyclotron Resonance): FT-ICR analyzers use magnetic fields to measure ion oscillation
frequencies and determine mass accurately.
Protein Sequencing Workflow via Mass Spectrometry
Sample Preparation:
The protein of interest is extracted and purified from a biological sample, such as cells or tissues. Sample
preparation may involve denaturation, reduction (breaking disulfide bonds), and alkylation (preventing disulfide
bond reformation). The protein is enzymatically digested into smaller peptides, typically using a proteolytic enzyme
like trypsin.
Mass Spectrometry Instrumentation:
Select an appropriate Mass Spectrometer, which can be MALDI-TOF, ESI-MS, Orbitrap, or FT-ICR, depending on the
specific requirements of the analysis. Choose between MALDI (for intact protein analysis) or ESI (for peptide
analysis) as the ionization source. The mass analyzer used depends on the instrument and can include TOF,
quadrupole, ion trap, Orbitrap, or FT-ICR.
Ionization:
In MALDI, a laser is used to ionize and desorb the sample from a matrix. In ESI, the sample is sprayed as a fine
mist, and the electric field at the tip of the spray capillary ionizes the molecules in solution.
Mass Analysis:
The mass spectrometer analyzes the ions produced from the ionization step. Ions are separated based on their
mass-to-charge ratio (m/z). A mass spectrum is generated, showing the intensity of ions at different m/z values.
Tandem Mass Spectrometry (MS/MS):
If protein sequencing or peptide identification is the goal, tandem MS is employed. Selective precursor ions are
fragmented in a collision cell. The resulting fragment ions are analyzed in a second mass analyzer to provide
sequence information.
Data Analysis:
Specialized software is used to interpret the mass spectra and identify peptide sequences. Database searching or de
novo sequencing algorithms are employed to match observed spectra to theoretical peptide sequences.
Post-translational modifications (PTMs) are identified by analyzing mass shifts in the spectra.
Protein Assembly:
Individual peptide sequences are assembled to reconstruct the complete protein sequence. Overlapping peptides provide
validation and ensure sequence accuracy.
Validation and Confirmation:
Experimental validation may be performed, such as manual inspection of spectra or additional MS/MS experiments. The
identified protein sequence is confirmed and reported.
Peptide identification in mass-spectrometry shotgun approach (Chugunova et al., 2018)
Mass Spectrometry Advantages in Protein Sequencing
Protein Identification:
Sensitivity: Mass spectrometry is highly sensitive and can detect proteins even at low concentrations within complex
mixtures. This sensitivity allows for the identification of trace amounts of proteins in biological samples.
Specificity: Mass spectrometry provides specific information about the mass-to-charge ratio of ions, enabling precise
protein identification. The comparison of experimental mass spectra with established protein sequence databases
ensures accurate identification.
Peptide Sequencing:
Accuracy: Tandem mass spectrometry (MS/MS or MS2) provides accurate sequencing of individual peptides. By fragmenting
peptides and analyzing the resulting fragment ion masses, the sequence of the peptide can be determined with a high
degree of confidence.
Coverage: MS/MS allows for the sequencing of multiple peptides within a protein, providing comprehensive coverage of
the protein's primary structure. This is especially valuable for proteins with large or complex sequences.
Post-Translational Modification
(PTM) Analysis:
Site-Specific Identification: Mass spectrometry can pinpoint the exact sites of PTMs on a protein. By measuring the
mass shifts of modified peptides, researchers can determine which amino acids have undergone modifications, offering
insights into protein function and regulation.
Multiplexing: Mass spectrometry can simultaneously analyze various PTMs on a single protein, allowing for the
comprehensive characterization of PTM profiles in complex biological systems.
De Novo Sequencing:
Independence from Databases: De novo sequencing using mass spectrometry does not rely on pre-existing protein
sequence databases. This makes it particularly useful for identifying novel proteins or proteins from organisms with
limited genomic information.
Sequencing Accuracy: Mass spectrometry can provide high sequencing accuracy in de novo mode, facilitating the
determination of amino acid sequences and, subsequently, the complete protein sequence.
Application of Mass Spectrometry in Protein Sequencing
Identification of Proteins: Mass spectrometry can be used to identify proteins in complex mixtures.
In a typical experiment, proteins are enzymatically digested into peptides, and these peptides are then ionized and
analyzed by mass spectrometry. The resulting mass spectra can be matched against protein sequence databases to
identify the proteins present in the sample.
De Novo Sequencing: Mass spectrometry can be employed for de novo sequencing of proteins. In this
approach, the mass spectrometer determines the mass-to-charge ratios of peptide fragments, and this information is
used to deduce the sequence of amino acids in the original protein. This is particularly valuable for analyzing
novel or uncharacterized proteins.
Post-translational Modification (PTM) Analysis: Proteins often undergo various PTMs, such as
phosphorylation, glycosylation, and acetylation, which play crucial roles in their functions. Mass spectrometry can
detect and localize these modifications by analyzing the mass shifts of peptides caused by PTMs.
Reference
- Chugunova, Anastasia, et al. "Mining for small translated ORFs." Journal of proteome research 17.1 (2018): 1-11.
For research use only, not intended for any clinical use.
