|
| 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 |
|
