Immobilized pH gradient-driven paper-based IEF: a new method for fractionating complex peptide mixtures before MS analysis

We present a method consisting of electrophoresis of complex mixtures of peptides using a strip of filter paper cut into 20 sections laid end to end over a 24-cm-long IPG strip, the pH gradient of which would drive the electrophoresis. Peptides absorbed onto individual paper pads after electrophoresis are subsequently recovered into a buffer solution, thus dividing a complex peptide mixture according to pI into 20 liquid fractions. This paper-based IEF method (PIEF) was compared side-by-side with a similar but liquid-based Offgel electrophoresis (OGE) by analyzing iTRAQ-labeled peptide mixtures of membrane proteins from four different cell types.PIEF outperformed OGE in resolving acidic peptides, whereas OGE did a better job in recovering relatively basic peptides. OGE and PIEF were quite comparable in their coverage, identifying almost equal number of distinct proteins (PIEF =1174; OGE = 1080). Interestingly, however, only 675 were identified by both of them, each method identifying many unique proteins (PIEF = 499; OGE = 415). Thus, the two methods uncovered almost 40% more proteins compared to what is obtained by only one method. Conclusion: This initial investigation demonstrates the technical feasibility of PIEF for complementing OGE. PIEF uses standard IPG IEF equipment, requires no specialized apparatus (e.g., OGE fractionator) and may be integrated into peptide mapping strategies for clinical samples.The complexity of the human proteome, in terms of its size (over 100,000 proteins/variants) and dynamic range (up to a billion-fold difference in abundance of the various types of proteins), is well recognized [1,2]. Because MS methods preferentially identify the most abundant proteins in complex mixtures [3], the ability to identify low-abundance proteins by MS requires application of a variety of pre-MS techniques for depleting and/or separating out abundant proteins [4-12] as well as techniques for fractionating peptides [13-17]. Of these techniques, Offgel e