Project PXD000314

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


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(s) : 24313913

Data Processing Protocol

See details in reference(s) : 24313913


Chia-Feng Tsai, Institute of Chemistry

Submission Date


Publication Date


Cell Type

B cell




Not available

Experiment Type

Bottom-up proteomics

Assay count



    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: 24313913


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 29920 Human lung cancer tissue 1104 5861 1577 36862 4828
2 29921 Human lung cancer tissue 858 4607 1209 33110 3576
3 29922 Raji B cell 678 2087 1068 15439 1981
4 29923 Raji B cell 663 2034 1045 15414 1909
5 29924 Raji B cell 661 2050 1045 14875 1929
6 29925 Raji B cell 677 2160 1077 15033 2014
7 29926 Raji B cell 647 2143 1059 15784 2041
8 29927 Raji B cell 660 2061 1037 15959 1948
9 29928 Raji B cell 634 2039 1011 15752 1917
10 29929 Raji B cell 663 2193 1062 15617 2058