Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins 185

Eilon Barnea, Raya Sorkin, TamarZiv, Ilan Beer and Arie Admon

8.1 Introduction 185

8.1.1 The HUPO Plasma Proteome Project (PPP) goals and the serum as a complex sample 185

8.1.2 The scope of this manuscript 187

8.2 Materials and methods 187

8.2.1 Depletion from serum albumin and antibodies 187

8.2.2 MudPITand mass segmentation 187

8.2.3 Protein separation by SDS-PAGE 188

8.2.4 SCX separation of intact proteins followed by MudPIT 188

8.2.5 Liquid-phase IEF followed by MudPIT 188

8.2.6 Capillary RP-LC-MS/MS 189

8.2.7 MS data processing and peptide/protein identifications 189

8.3 Results 189

8.3.1 Comparisons between the prefractionation methods 190

8.3.2 Identification of different protein subsets 191

8.3.3 Proteins identified by only one prefractionation method 193

8.3.4 Different methods resulted in diverse peptide coverage 193

8.4 Discussion 196

8.4.1 Giving every peptide a chance 196

8.4.2 How to identify more of the marginal proteins 197

8.4.3 Clustering and comparing raw data 197

8.4.4 High throughput and ruggedness versus high sensitivity 197

8.4.5 The cost effectiveness of the different methods 198

8.5 Concluding remarks 198

8.6 References 199

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