Table 5 Comparison of the depletion and enrichment approaches to combinatorial peptidomics

1. Individual steps per each "filtering" stage

1 step process, i.e. bind to beads (beads discarded)

3 step process, i.e. bind to beads, wash and elute

2. Number of combinations using 6 amino acid filters

63

63

3. Range of suitable peptide lengths

Longer peptides require less "filtering" stages (i.e. 10 or more amino acid residues preferred)

Shorter peptides require less "filtering" stages (i.e. 10 or less amino acid residues preferred)

4. Complexity of peptide mixtures

Decreased

Decreased

5. Amino acid compositional complexity of the remaining peptides

Decreased (by the number of "filters" used)

Not changed (20 amino acids)

6. Quantitative analysis

A single-stage depletion is more straightforward and quantitative than a triple-stage enrichment

Enrichment approach is less straightforward and robust than the depletion

7. Scaling up

Possible (larger "filters" or consecutive stages)

Possible (larger "filters" or parallel reactions)

8. Scaling down

Possible (low fmol level MS sensitivity requires high pmol filter binding capacities)

Especially suitable : low fmol level MS sensitivity requires fmol binding capacities

9A. Limitations (overloading)

Large binding capacity of the "filters" is crucial – overloading will allow all peptides to pass the "filter"

Overloading of the "filters" is not an issue, excess of sample may be applied

9A. Limitations (incompletely digestion)

Products of incomplete digestion will be mostly eliminated

Products of incomplete digestion will be mostly retained and may interfere with the downstream purification and analysis steps

10. Nano-applications

Problematic due to limitation (see above) – excess of binding sites required to maintain efficient separation. Suitable for micro-fluidic applications

Suitable for nano-applications, since smaller number of binding sites required (compared to depletion strategy)