Peptide Sequencing & Proteomics – Core Vocabulary

Vocabulary flashcards covering fundamental terms, instruments, techniques and quantitative strategies involved in mass-spectrometry-based peptide sequencing and proteomics.

Edman degradation

Classical chemical method for sequencing proteins by stepwise removal and identification of N-terminal amino acids; requires a free amino terminus and large amounts of purified protein.

Mass spectrometry (MS)

Analytical technique that ionizes molecules and measures their mass-to-charge (m/z) ratios to identify and characterize biomolecules such as peptides and proteins.

Peptide sequencing

Determining the amino-acid order in a peptide, typically by analysing fragmentation patterns generated during tandem mass spectrometry.

Proteomics

Large-scale study of the full complement of proteins (proteome) in a cell, tissue or organism, often using mass-spectrometry-based methods.

Electrospray ionization (ESI)

‘Soft’ ionization technique in which a high-voltage needle turns liquid effluent into charged droplets that evaporate to release multiply charged peptide ions.

Matrix-assisted laser desorption/ionization (MALDI)

Ionization method where analytes mixed with a light-absorbing matrix are desorbed and ionized by a laser pulse, usually producing singly charged ions.

Microscale capillary HPLC column

Very narrow reversed-phase chromatography column (50–150 µm i.d.) used to separate peptide mixtures at nanolitre flow rates prior to MS.

m/z ratio

Mass-to-charge value measured by a mass spectrometer; peptide mass divided by the number of charges.

Quadrupole mass spectrometer

Mass analyser that filters ions by stabilizing specific m/z trajectories through four parallel rods with oscillating electric fields.

Time-of-flight (TOF) mass spectrometer

Instrument that separates ions by measuring the time they need to travel a field-free flight tube; lighter ions arrive sooner than heavier ones.

Ion trap (quadrupole ion trap)

Mass analyser that traps ions in a dynamic electric field, then ejects them sequentially by m/z; enables MSn (multiple rounds of fragmentation).

Fourier-transform ion cyclotron resonance (FTICR-MS)

High-resolution mass analyser where ions orbit in a strong magnetic field; image currents are converted via Fourier transformation to yield m/z values with ppm accuracy.

Tandem MS (MS/MS)

Two-stage experiment in which a selected precursor ion is fragmented, and the masses of product ions are measured to obtain sequence information.

Precursor ion

Selected peptide ion that is isolated for fragmentation in a tandem-MS experiment.

Product (fragment) ions

Ions generated after fragmentation of the precursor during tandem MS; used to deduce peptide sequence.

b-ion

Fragment retaining the peptide’s N-terminal charge after cleavage at the peptide bond during collision-induced dissociation.

y-ion

Fragment retaining the peptide’s C-terminal charge after cleavage at the peptide bond; often dominant in quadrupole and TOF spectra.

De novo sequencing

Deriving a peptide’s amino-acid sequence directly from its MS/MS spectrum without referencing a database.

Peptide-sequence tag

Short, confidently assigned amino-acid string plus adjacent mass values used to match MS/MS data to database sequences.

Database searching (e.g., Sequest, Mascot)

Bioinformatic comparison of experimental MS/MS spectra with theoretical peptide fragments from protein databases to identify proteins.

False positive

Protein or peptide identification reported by software that is actually incorrect; arises from random spectral matches.

Total ion current (TIC)

Sum of all ion intensities recorded in each mass spectrum plotted over chromatography time.

Extracted ion chromatogram (XIC)

Plot showing the signal intensity of one specific m/z value across the chromatographic run, used for quantification.

Ionization efficiency

Fraction of molecules in solution that become gas-phase ions; differs between peptides and influences signal intensity.

Trypsin

Highly specific protease that cleaves peptide bonds C-terminal to lysine and arginine, generating MS-friendly peptides (~0–20 aa).

Lys-C

Protease that cleaves C-terminal to lysine; stable in harsh conditions and often used before trypsin digestion.

MudPIT (multidimensional protein identification technology)

Shotgun proteomics workflow combining strong-cation-exchange and reversed-phase LC with tandem MS for large-scale protein identification.

GeLC-MS

Workflow in which proteins separated by SDS–PAGE are excised, digested in-gel, and analysed by LC–MS/MS slice by slice.

Peptide mass fingerprinting

Protein identification by matching a set of peptide masses (usually MALDI) to theoretical tryptic masses in a database.

Top-down sequencing

Approach that analyses intact proteins (rather than digested peptides) in the mass spectrometer to obtain sequence and modification information.

Stable-isotope labelling by amino acids in cell culture (SILAC)

Metabolic labelling method where cells incorporate heavy isotope-labelled amino acids, enabling accurate MS-based quantification between states.

Isotope-coded affinity tag (ICAT)

Chemical label consisting of a cysteine-reactive group, isotopically light or heavy linker, and biotin; allows enrichment and relative quantification of cysteine-containing peptides.

Post-harvest labelling

Introducing stable-isotope tags chemically after cell lysis/protein extraction, enabling quantitative MS when metabolic labelling is impossible.

Absolute quantification (AQUA)

Method that spikes known amounts of stable-isotope-labelled synthetic peptides into samples to determine absolute amounts of target proteins.

Protein correlation profiling

Strategy that distinguishes true organelle or complex components from contaminants by comparing their fractionation patterns across gradients.

Shotgun proteomics

MS approach that digests complex protein mixtures into peptides and analyses them directly without prior protein separation.

Surface-enhanced laser desorption/ionization (SELDI)

MALDI variant where proteins bind to chemically modified surfaces before laser desorption, generating diagnostic spectral patterns.

Hydrophobicity gradient (in RP-HPLC)

Solvent composition increasing in organic content to elute peptides from a reversed-phase column in order of hydrophobicity.

Charge state

Number of positive charges carried by an ion; derived from the 1 Da spacing in isotopic clusters of peptide peaks.

Collision-induced dissociation (CID)

Fragmentation process where ions collide with inert gas molecules, gaining energy that breaks peptide bonds to generate MS/MS spectra.

Immonium ion

Low-mass fragment representing a single amino acid’s side-chain structure; useful as diagnostic marker for specific modifications (e.g., phosphotyrosine).