A novel fourdimensional strategy combining protein and peptide separation methods enables detection of lowabundance proteins in human plasma and serum proteomes 135

Hsin-Yao Tang, Nadeem Ali-Khan, Lynn A. Echan, Natasha Levenkova, John J. Rux and David W. Speicher

6.1 Introduction 135

6.2 Materials and methods 138

6.2.1 Materials 138

6.2.2 Top six protein depletion 138

6.2.3 MicroSol-IEF fractionation 139

6.2.4 Protein array pixelation 139

6.2.5 LC-ESI-MS/MS methods 140

6.2.6 Data analysis 140

6.3 Results and discussion 141

6.3.1 Protein array pixelation strategy 141

6.3.2 Optimization of protein array pixelation 143

6.3.3 Total analysis time for protein array pixelation of human plasma proteome 146

6.3.4 Systematic protein array pixelation of the human plasma proteome 147

6.3.5 Systematic protein array pixelation of the human serum proteome 150

6.3.6 Analyses of human plasma and serum proteomes using HUPO filter criteria 153

6.4 Concluding remarks 157

6.5 References 157

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