Project : PXD000314

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Summary

Title

Sequential Phosphoproteomic Enrichment by Complementary Metal-directed Immobilized Metal Ion Affinity Chromatography: Case Study on Kinase Substrate Mapping in Human Lung Cancer Tissue

Description

Abstract: Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, methodologies to enrich the heterogeneous types of phosphopeptides are critical towards comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinctive binding affinity of Ga3+ and Fe3+ towards phosphopeptides, we designed a tip-based metal-directed immobilized metal ion affinity (MD-IMAC) chromatography for sequential enrichment of phosphopeptides. On the analysis of Raji B cell, this sequential Ga3+-Fe3+-IMAC strategy demonstrated 1.5-3.5 fold superior phosphoproteomic coverage compared to the single IMAC (Fe3+, Ti4+, Ga3+ and Al3+). In addition, as high as 92% among the 6283 phosphopeptides was uniquely enriched by either 1st Ga3+-IMAC fraction (41%) or 2nd Fe3+-IMAC fraction (51%). The complementary property of Ga3+ and Fe3+ was further shown on the exclusively superior efficiency to enriched almost all the 1214 multiply phosphorylated peptides (99.4%) by 1st Ga3+-IMAC, while as low as 10% of 5069 monophosphorylated phosphopeptides was commonly enriched by both fractions. Application of our sequential Ga3+-Fe3+-IMAC approach to a human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlapping. The fractionation ability was shown not only on the mono phosphopeptides and multiply phosphopeptides but also on the basic and acidic phosphopeptides; acidiphilic phosphorylation sites were predominately present in 1st Ga3+-IMAC (72%) and 85% Pro-directed and 79% basophilic phosphorylation sites were enriched by 2nd Fe3+-IMAC. Most interestingly, this strategy complementarily mapped different kinase substrate on the protein as well as site levels in multiple cellular pathways related to cancer invasion and metastasis of lung cancer ., Given the demonstrated fractionation ability, reproducibility, sensitivity and ease of tip preparation, we hope that this Ga3+-Fe3+-IMAC allow more comprehensive characterization of phosphoproteome in vitro and in vivo. Database search: The raw MS/MS data obtained by TripleTOF 5600 were processed using AB_SCIEX MS Data Converter with default parameters. All MS/MS files were analyzed using Mascot (Matrix Science, London, UK; version 2.3) against the SwissPort database (version 57.8) with the following constraints: an allowance for tryptic peptides of up to two missed cleavage sites, a fragment ion mass tolerance of 0.05 Da, and a parent ion tolerance of 10 ppm. Phosphorylation (S, T, Y) and oxidation (M) were selected as variable modifications. Searching on a randomized decoy database created by Mascot was required to evaluate the false discovery rate associated with protein identification. The false discovery rates with a Mascot score (p< 0.05) ranged between 0% and 1% in this study.

Sample Processing Protocol

See details in reference PMID : 24313913

Data Processing Protocol

See details in reference PMID : 24313913

Contact

Chia-Feng Tsai, Institute of Chemistry

Submission Date

02/07/2013

Cell Type

B cell

Instrument

TT5600

Quantification

Not available

Experiment Type

Bottom-up proteomics

Assay count

29

Publication

    Tsai CF, Hsu CC, Hung JN, Wang YT, Choong WK, Zeng MY, Lin PY, Hong RW, Sung TY, Chen YJ. Sequential Phosphoproteomic Enrichment through Complementary Metal-Directed Immobilized Metal Ion Affinity Chromatography. Anal Chem. 2014 Jan 7;86(1):685-93 PubMed(s) : 24313913

Assay

Accession Title Protein Count Peptide Count Unique Peptide Count Spectrum Count Identified Spectrum Count
29920 Human lung cancer tissue 1104 5861 1577 36862 4828
29921 Human lung cancer tissue 858 4607 1209 33110 3576
29922 Raji B cell 678 2087 1068 15439 1981
29923 Raji B cell 663 2034 1045 15414 1909
29924 Raji B cell 661 2050 1045 14875 1929
29925 Raji B cell 677 2160 1077 15033 2014
29926 Raji B cell 647 2143 1059 15784 2041
29927 Raji B cell 660 2061 1037 15959 1948
29928 Raji B cell 634 2039 1011 15752 1917
29929 Raji B cell 663 2193 1062 15617 2058
29930 Raji B cell 643 2034 1018 15555 1932
29931 Raji B cell 969 2832 1462 14609 2635
29932 Raji B cell 919 2766 1412 14062 2591
29933 Raji B cell 934 2652 1394 14095 2504
29934 Raji B cell 888 2515 1332 14041 2329
29935 Raji B cell 913 2578 1356 13855 2423
29936 Raji B cell 891 2502 1345 13756 2381
29937 Raji B cell 887 2700 1460 18782 2597
29938 Raji B cell 956 2960 1526 18887 2789
29939 Raji B cell 930 2903 1519 18982 2742
29940 Raji B cell 959 2885 1537 19044 2766
29941 Raji B cell 983 3155 1649 19307 2994
29942 Raji B cell 1029 3300 1731 19174 3145
29943 Raji B cell 1059 3425 1736 19130 3187
29944 Raji B cell 991 3206 1670 19229 3011
29945 Raji B cell 1023 3272 1718 19426 3070
29946 Raji B cell 870 2386 1286 13701 2265
29947 Raji B cell 906 2553 1352 13826 2391
29948 Raji B cell 906 2612 1335 13590 2415

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