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ZIC-cHILIC as a fractionation method for sensitive and powerful shotgun proteomics

Nature Protocols volume 7, pages 2041–2055 (2012)Cite this article

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Abstract

Multidimensional liquid chromatography (LC) combined with mass spectrometry (MS) has become a standard technique in proteomics to reduce sample complexity and to tackle the dynamic range in protein abundance. Fractionation is necessary to obtain a comprehensive analysis of complex biological samples such as tissue and mammalian cell lines. However, extensive fractionation comes at the expense of sample loss, presenting a bottleneck in the analysis of limited amounts of material. In this protocol, we describe a two-dimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography (HILIC; with a zwitterionic packing material, ZIC-cHILIC) and reversed-phase chromatography, which allows proteomic analyses with minimal sample loss. Experimental aspects related to obtaining maximum recovery are discussed, including how to optimally prepare samples for this system. Examples involving protein lysates originating from cultured cell lines and cells sorted by flow cytometry are used to show the power, sensitivity and versatility of the technique. Once the ZIC-cHILIC fractionation system has been optimized and standardized, this protocol requires ∼5–6 d, including sample preparation and fraction analysis.

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Figure 1: Schematic representation of the nanoflow ZIC-cHILIC system.
Figure 2: Schematic representation of the fused silica capillaries during different steps of the packing procedure.
Figure 3: Schematic design of a two-dimensional HILIC-RP approach.
Figure 4: Experimental setups.
Figure 5: LC-MS chromatograms for the analysis of 1 pmol of a standard peptide mixture (BSA, α-casein and β-casein) using the ZIC-cHILIC configuration.
Figure 6: Fractional distributions of unique peptides from a HeLa digest after ZIC-cHILIC fractionation.

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Acknowledgements

We thank P.J. Boersema and R. Raijmakers for suggestions and critical reading of the manuscript. We thank all members of the Biomolecular Mass Spectrometry and Proteomics Group, particularly H. Post, A. Barendregt and S. Goerdayal, for their help. This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union Seventh Framework Program. The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative and The Netherlands Organization for Scientific Research (NWO) with the VIDI grant 700.10.429 (for S.M.), is acknowledged for funding.

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  1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands

    Serena Di Palma, Shabaz Mohammed & Albert J R Heck

  2. Netherlands Proteomics Centre, Utrecht, The Netherlands

    Serena Di Palma, Shabaz Mohammed & Albert J R Heck

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  1. Serena Di Palma
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  2. Shabaz Mohammed
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S.D.P. designed and performed the experiments, analyzed data and wrote the paper. S.M. and A.J.R.H. designed the experiments, supervised the project and wrote the paper.

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Correspondence to Shabaz Mohammed or Albert J R Heck.

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Alternative sample preparation procedure for larger amounts of starting material (PDF 395 kb)

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Di Palma, S., Mohammed, S. & Heck, A. ZIC-cHILIC as a fractionation method for sensitive and powerful shotgun proteomics. Nat Protoc 7, 2041–2055 (2012). https://doi.org/10.1038/nprot.2012.124

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