Proteome Project reference specimens Systematic variation between sample types and calibration of mass spectrometry data

Brian B. Haab, Bernhard H. Geierstanger, George Michailidis, Frank Vitzthum, Sara Forrester, Ryan Okon, Petri Saviranta, Achim Brinker, Martin Sorette, Lorah Perlee, Shubha Suresh, Garry Drwal, Joshua N. Adkins and Gilbert S. Omenn

4.1 Introduction 92

4.2 Materials and methods 93

4.2.1 Reference specimens 93

4.2.2 DB immunoassays 93

4.2.3 Antibody arrays at GNF 94

4.2.3.1 Antibodies, reagents, microarray printing, and platform 94

4.2.3.2 Microarray layout and processing 94

4.2.3.3 Array imaging and data analysis 95

4.2.4 Antibody microarrays at MSI 95

4.2.4.1 Chip manufacture 95

4.2.4.2 Rolling circle amplification (RCA) immunoassay 96

4.2.4.3 Conversion ofmean fluorescent intensity to concentration 96

4.2.5 Antibody microarrays at VARI 96

4.2.5.1 Fabrication of antibody microarrays 96

4.2.5.2 Serum labeling 97

4.2.5.3 Processing of antibody microarrays 97

4.2.5.4 Analysis 97

4.2.6 Retrieval and matching of IPI numbers for the analytes 97

4.3 Results 98

4.3.1 Antibody-based measurements of the HUPO reference specimens 98

4.3.2 Systematic variation between the preparation methods ofthe PPP reference specimens 200

4.3.3 Consistent alterations in specific protein abundances 207

4.3.4 Linkage of MS data and antibody-based measurements 208

4.4 Discussion 220

4.5 References 223

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