Evaluation of Enrichment Techniques for Mass Spectrometry Jonathan A. Schumacher, David K. Crockett, Kojo S.J. Elenitoba-Johnson, Megan S. Lim The Journal of Molecular Diagnostics Volume 9, Issue 2, Pages 169-177 (April 2007) DOI: 10.2353/jmoldx.2007.060031 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 1 Tyrosine phosphoproteins from SU-DHL-1 cells were enriched by immunoprecipitation (left) or immunoaffinity chromatography (right). Subsequently, enriched tyrosine phosphoproteins were subjected to immunoblot, silver stain, tryptic digestion, and LC-MS/MS analysis. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 2 Effect of sodium orthovanadate on tyrosine phosphoproteins. Lysates from SU-DHL-1 cells treated (+) or untreated (−) with sodium orthovanadate were immunoprecipitated and probed with the 4G10 anti-phosphotyrosine antibody. The lane corresponding to sodium orthovanadate-treated cells illustrates a total of 16 immunoreactive bands ranging from 130 through 8 kd. The lane corresponding to untreated cells illustrates a total of nine immunoreactive bands ranging from 130 through 8 kd. Densitometry analysis of the band corresponding to NPM-ALK, denoted by arrows, demonstrated an 18% increase in band intensity in the lane corresponding to sodium orthovanadate-treated cells. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 3 4G10 and sc-18182 enrich for tyrosine phosphoproteins. Lysates from SU-DHL-1 cells treated with 2 mmol/L sodium orthovanadate were immunoprecipitated and probed using sc-18182 (left) or 4G10 (right). The lane corresponding to immunoprecipitation using sc-18182 demonstrates 14 immunoreactive bands ranging in size from 4 to 130 kd. The lane corresponding to proteins enriched by immunoprecipitation using 4G10 illustrates a total of 15 immunoreactive bands ranging from 8 to 120 kd. Densitometry analysis of the bands corresponding to NPM-ALK (arrow) demonstrated a 22% increase in intensity in the lane corresponding to tyrosine phosphoproteins immunoprecipitated with 4G10. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 4 Enrichment of tyrosine phosphoproteins by immunoaffinity chromatography and immunoprecipitation and confirmation of equal loading. Tyrosine phosphoproteins from SU-DHL-1 cells were enriched by immunoaffinity chromatography or immunoprecipitation and probed with the 4G10 antibody. A: The lane corresponding to immunoaffinity chromatography (IA) demonstrates 10 immunoreactive bands ranging from 8 to 120 kd. In contrast, the lane corresponding to immunoprecipitation (IP) demonstrates 17 immunoreactive bands ranging from 8 to 120 kd. Densitometry analysis of the band corresponding to NPM-ALK, denoted by arrows, demonstrated a 44% increase in band intensity in the lane corresponding to proteins enriched by immunoprecipitation over immunoaffinity chromatography. B: A Coomassie blue-stained gel of tyrosine phosphoproteins enriched by immunoaffinity chromatography (IA) or immunoprecipitation (IP). Gel demonstrates equal loading between the two samples. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 5 Identification of ETK tyrosine kinase by LC-MS/MS. MS full scan and data-dependent MS/MS scan identified the tryptic peptide QPYDIpYDNSQV. Peptide sequencing is indicated by the B-ion (red) and Y-ion (blue) fragment series, with the identified peptide matching to ETK in the electronic database search. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 6 Analysis of cellular distribution of identified proteins enriched for by immunoprecipitation (A) and immunoaffinity chromatography (B). SU-DHL-1 cells were grown to 8 × 105 cells/ml and protein precipitated as described in Materials and Methods. Immunoprecipitation, SDS-PAGE, silver stain, and LC-MS/MS were performed as described. Proteins were analyzed for cellular distribution using GOMiner. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions
Figure 7 Analysis of molecular functions of identified proteins enriched for by immunoprecipitation (A) and immunoaffinity chromatography (B). SU-DHL-1 cells were grown to 8 × 105 cells/ml and protein precipitated as described in Materials and Methods. Immunoprecipitation, SDS-PAGE, silver stain, and LC-MS/MS were performed as described. Proteins were analyzed for molecular function using GOMiner. The Journal of Molecular Diagnostics 2007 9, 169-177DOI: (10.2353/jmoldx.2007.060031) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions