Table 1.
Summary of the strengths and weaknesses of analytical tools used in peptidome research as discussed in this review. Abbreviations: IEX: ion exchange; LC: liquid chromatography; MWCO: molecular weight cut-off; OS: organic solvent extraction; PAGE: polyacrylamide gel electrophoresis; RAM: restricted access material; RP: reversed phase; SPE: solid phase extraction; UF: ultrafiltration; SEC: size exclusion chromatography.
| Technique | Strengths | Weaknesses |
|---|---|---|
| Depletion | (i) Removes highly abundant “household” proteins, allowing a “deeper” look into the peptidome | (i) Requires costly antibody columns |
| (ii) Each protein to be removed requires a different specific antibody | ||
| (iii) Loss of peptides by nonspecific binding | ||
| PAGE | (i) Traditional well-established method (ii) able to separate isoforms or PTMs (iii) able to remove low molecular organic and inorganic impurities |
(i) Unsuitable for highly complex samples, poor dynamic range (ii) Poor resolving power (iii) Proteins/peptides with extreme pI values cannot be separated (iv) The smallest peptides are not retained in MW separation dimension (v) Very time consuming and laborious (vi) Low dynamic range |
| OFFGel | (i) Effective prefractionation tool (ii) Medium resolution |
(i) Postconcentration is required low resolution (ii) No MWCO discrimination (iii) long separation times |
| UF | (i) Fast (ii) Inexpensive (iii) Easy to automate |
(i) Variable quality and reproducibility of commercial devices (ii) Loss of hydrophobic peptides by nonspecific binding |
| LC (SPE/RP/IEX) |
(i) High resolution (ii) Directly compatible with MS (iii) High sensitivity in nanoLC (iv) SPE flexible, easy to automate (v) Efficient preconcentration |
(i) Extensive method development for each specific matrix is required |
| SEC | (i) High resolving power (ii) Reproducible |
(i) Loading limited by small injection volume |
| RAM | (i) Effective removal of HMW compounds (ii) Relatively large injection volumes (iii) Analysis of untreated sample matrices possible (iv) Easy to automate (v) Online RAM coupled to (nano-)LC-MS/MS possible |
(i) Complicated LC setup (ii) Extensive application directed method development and optimization required |
| OS | (i) Easy to operate (ii) Inexpensive |
(i) Tedious to perform |