Table 1 Summary of proteomics and chromatography techniques used in quantitative and qualitative proteomics
Technique | Details | Application | Strengths | Limitations |
|---|---|---|---|---|
iTRAQ | Isobaric tags for relative and absolute quantitation | • Protein quantification through incorporation of stable isotopes • Isobaric tagging of peptides | • Multiplex several samples • Relative quantification • High-throughput | • Increases sample complexity • Require fractionation of peptides before MS |
SILAC | Stable isotope labeling with amino acids in cell culture | • SILAC relies on metabolic incorporation of a given ‘light’ or ‘heavy’ form of the amino acid into the proteins, • Direct isotope labeling of cells Differential expression pattern | • Degree of labeling is significantly high • Quantitation is straightforward | • SILAC labeling of tissue samples is not possible |
ICAT | Isotope-coded affinity tag | • Chemical isotope labeling for quantitative proteomics | Sensitive and reproducible Detect peptides with low expression levels | • Proteins without cysteine residues and acidic proteins are not detected |
TMT | Tandem Mass Tag | • Protein quantification through incorporation of stable isotopes • Isobaric tagging of peptides | • Identification and quantitation of proteins in different samples • Relative quantification • Targeted quantitation strategies like SRM • High-throughput | • Increases sample complexity • Require fractionation of peptides before MS |
HILIC | Hydrophilic Interaction Liquid Chromatography | • Analysis of charged substances • Separating polar proteins\peptides • Separation and quantitative analysis of modified and unmodified peptides | • The altered charge-state and hydrophilicities of the DPM-modified peptides make it possible to distinguish these from their unmodified counterparts via LC-MS/MS | • Longer column equilibration time, • Less reproducible retention times, • Higher cost of mobile phase |
emHILIC | Electrostatic-interaction Modified HILIC hydrophilic interaction liquid chromatography | • Separation and quantitative analysis of modified peptides | • Efficient separation of modified peptides from unmodified via LC-MS/MS | • Some peptides may not dissolve well in high organic solvent (90%ACN) |
ERLIC using WAX or SAX | Electrostatic-Repulsion Hydrophilic Interaction chromatography | • Separation of isoforms of peptides and proteins based on pI and hyrophobicities. • Study protein DPMs/PTMs to inferior their biological functions based on quantitation | • Quantitation of isoforms of peptides and proteins, e.g. the trios of deamidation products. | • Some peptides may not dissolve well in high organic solvent (90%ACN) • ERLIC chromatographic resolution is lower than C18 RP column. |
LERIC-MS/MS | Long-length Column Electrostatic-Repulsion Hydrophilic Interaction chromatography coupled to tandem MS | • Study global protein DPMs/PTMs in whole complex proteomes like brain tissue lysate or cell lysate. | • Record the whole proteome in complex sample in a single LC-MS/MS data file for global DPMs/PTMs analysis. | • Some peptides may not dissolve well in high organic solvent (90%ACN). • ERLIC chromatographic resolution is lower than C18 RP column. |