I’m personally quite surprised that you can’t identify a band from a gel, that should be straight forward. I generally find if you can see a CBB G250 stained band you’ve got an excellent chance of identifying that protein or proteins, especially using LC-MS/MS.

That said, you’ve obviously got to tailor your purification and digestion regime to the proteins in which you have the most interest (which it sounds like you have done here). Membrane proteins are particularly difficult to work with, but there are new papers out all the time improving their characterisation using proteomics.

Do a search, see what techniques are suitable for your particular area, some to get you started;

M. Antelo-Varela, J. Bartel, A. Quesada-Ganuza, K. Appel, M. Bernal-Cabas, T. Sura, A. Otto, M. Rasmussen, J.M. van Dijl, A. Nielsen, S. Maaß, D. Becher, Ariadne’s Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins, Anal. Chem. (2019). https://doi.org/10.1021/acs.analchem.9b02869.

J. Loraine, O. Alhumaidan, A.R. Bottrill, S.C. Mistry, P. Andrew, G.V. Mukamolova, O. Turapov, Efficient Protein Digestion at Elevated Temperature in the Presence of Sodium Dodecyl Sulfate and Calcium Ions for Membrane Proteomics, Anal. Chem. (2019). https://doi.org/10.1021/acs.analchem.9b00484.

O. Vit, K. Harant, P. Klener, P. Man, Petrak, A three-pronged “Pitchfork” strategy enables an extensive description of the human membrane proteome and the identification of missing proteins, Journal of Proteomics. (2019) 103411. https://doi.org/10.1016/j.jprot.2019.103411.

Y. Zhang, Z. Lin, P. Hao, K. Hou, Y. Sui, K. Zhang, Y. He, H. Li, H. Yang, S. Liu, Y. Ren, Improvement of Peptide Separation for Exploring the Missing Proteins Localised on Membranes, J. Proteome Res. (2018). https://doi.org/10.1021/acs.jproteome.8b00409.

M. Weldemariam, C.-L. Han, F. Shekari, R.B. Kitata, C.-Y. Chuang, W.-T. Hsu, H.-C. Kuo, W.-K. Choong, T.-Y. Sung, F.-C. He, M.C.M. Chung, G.H. Salekdeh, Y.-J. Chen, Subcellular Proteome Landscape of Human Embryonic Stem Cells Revealed Missing Membrane Proteins, J. Proteome Res. (2018). https://doi.org/10.1021/acs.jproteome.8b00407.

Y. Zhou, J. Gao, H. Zhu, J. Xu, H. He, L. Gu, H. Wang, J. Chen, D. Ma, H. Zhou, J. Zheng, Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach, Anal. Chem. (2018). https://doi.org/10.1021/acs.analchem.7b03710.

S. Hu, L. Musante, D. Tataruch, X. Xu, O. Kretz, M. Henry, P. Meleady, H. Luo, H. Zou, Y. Jiang, H. Holthofer, Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning, J. Proteome Res. (2017). https://doi.org/10.1021/acs.jproteome.7b00386.

Q. Zhao, F. Fang, Y. Shan, Z. Sui, B. Zhao, Z. Liang, L. Zhang, Y. Zhang, In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis, Anal. Chem. (2017). https://doi.org/10.1021/acs.analchem.6b04232.

Hi Thomas,

although there is certainly a bias, in general the strategy with trypsin digestion is a good choice for the average protein. Unfortunately, your protein is not an average protein.

When you have the protein as a band in an SDS-PAGE, you already have overcome a lot of the problems you could possibly have with membrane proteins.

The issues left are the reduced number of cleavage sites and the availability of these sites. To overcome the number of cleavage sites you can use different enzymes for the digest. Here a combination of trypsin with chymotrypsin is a good choice. The second problem is the availability of the cleavage sites. Here you can add methanol or ACN to your tryptic digest solution. Trypsin and Chymotrypsin will tolerate up to 60% of organic in the buffer during the digest without loss of specificity and activity. Here you might go with 40% ACN for appropriate results.

An additional problem might the size of the protein. If it is very small, you might lose it, during the pretreatment of the band prior to the digest. The destaining solutions may elute a small hydrophobic protein from the gel. As an alternative to Coomassie stain you might use an MS compatible silver staining. The destaining just requires a view minutes and works well with membrane proteins.

It might also be worth thinking of Edman degradation. Maybe not the most popular method. It works well and the machines are widespread.

Good luck!

As you are able to get it in solution to load on the gel, that's a lot of headache overcome, as Sascha Rexroth also noted. I assume you cut out the band and attempt in-gel digestion?

Alternative approaches could also be GELFrEE pre-fractionation or electroelution from the gel followed by in-solution digest with SDC as detergent (i have also seen a detergent called RapiGest that should work well with membrane proteins) and addition of some organic solvent as also noted above. Naturally, you also have to take protease specificity into the equation. One alternative option to chymotrypsin could also be to use limitied digestion with low-specificity proteases (perhaps thermolysin - however peptides may be too small?) followed by e.g. a non-specific search in MaxQuant. Naturally, a lot of quantitative information will be lost, but it appears that identification is of highest imprtance. You could also do intact protein MS on the eluted band to get an idea about exact MW of your protein and use that to curate your reference proteome, do in silico digestion, and setup a targeted DIA experiment on the MS. That could hopefully pick up some of the tryptic peptides by removing noise in the MS2 isolation.

I wish you the best of luck - sound like a bit tricky problem. Until you find the approach that work, naturally.