Table 1.

A how-to guide for MS-based proteomic characterization of viral infection

How to…	Technique	Description	Advantages	Drawbacks
Qualitative
Confirm viral protein expression or purity from low-complexity samples	Mass fingerprint	MALDI-TOF detection of intact peptide masses (MS¹); can be preceded by one- or two-dimensional electrophoresis	Rapid protein identification	Requires low protein complexity; limited sequence validation
Characterize viral and host proteomes	Peptide sequencing (tandem MS)	Predominant technology for viral proteomic studies; determination of peptide amino acid sequences using gas-phase fragmentation	Minimal sample handling and deep proteome coverage	Specialized computational approaches needed for large data sets
Quantitative
Identify differences in relative protein expression between mock- and virus-infected conditions	Spectral counting (label-free)	Relative abundance differences inferred by comparing number of spectra per protein acquired by tandem MS	Facile; no modification to qualitative workflows	Subtle expression differences not detected
Quantify relative protein expression between different mock- and virus-infected conditions	Peak area (label-free)	Intact peptide abundances are integrated by area under the curve; relative or absolute (with external calibration) quantification	Greater dynamic range and precision than spectral counting	High-resolution instrumentation needed; computationally demanding
Quantify relative protein expression and posttranslational modifications (usually between two or three cellular conditions)	Metabolic labeling	Protein labeling in cells or animals with light or heavy isotopes (e.g., ¹⁵N salts or ¹³C/¹⁵N amino acids); relative peak areas of light and heavy peptides in MS¹ spectra are used for quantification	Minimizes experimental variation; accurate measurement of small changes in relative expression	Increased cost, spectrum complexity, and computational demands; limited/expensive use in animals
Simultaneously quantify relative protein expression at different time points postinfection	Isobaric tagging	Peptide labeling (following digestion) using amine-reactive reagents; simultaneous relative quantification of up to 10 samples	High-throughput multiplexing of time points or biological replicates	Reduced linear quantification range
Determine the absolute abundance of proteins in virus-infected cells	Selected reaction monitoring (SRM)	Targeted analysis of selected peptides (e.g., <100) by tandem MS for relative or absolute quantification; often paired with isotope-labeled peptide/protein standards (e.g., AQUA or QconCAT)	Higher sensitivity and quantitative precision (<10%) than MS¹-based methods; identified signature peptides are universal among labs	Each method requires separate optimization and validation
Determine viral capsid assembly states and subunit arrangements under different salt and pH conditions	Native ion mobility MS	Analysis of intact proteins or noncovalent protein assemblies at lower complexity using a high-resolution mass detector (e.g., TOF or Orbitrap); ion mobility separates by structural “compactness”	Distinguish among structural isoforms of large protein complexes	Currently requires micromolar concentrations; technically challenging
Determine interprotein contacts within capsids or other complexes; map immune cell epitopes for antivirals and vaccines	Hydrogen/deuterium exchange	Quantify the differential uptake or release of deuterium from amide hydrogen depending on solvent accessibility of a protein surface and hydrogen bonding	Detect shifts in protein conformations; resolution of several amino acids	Technically challenging; back-exchange and bias toward fast-exchanging regions
Determine protein interaction topologies in a cellular context	Cross-linking MS (XL-MS)	Covalent linking of proteins; stabilizes weak interactions in AP-MS; exact cross-linked residues identified by tandem MS	Determine direct interactions at single-residue resolution	Cross-linked species are of low abundance; technically challenging

Abbreviations: AP, affinity purification; AQUA, absolute quantification; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; QConCAT, quantification concatemer; TOF, time of flight.