You SHOULD report all the proteins from a spot; as everyone here has noted, it's more-or-less expected that each spot probably represents more than one protein. With respect to what characteristics you should report: in general the more peptides with high-quality data (either exact masses or good CID spectra, the better. Three is probably a general minimum, but a single UNIQUE peptide with especially good data is sort-of OK if you don't over-interpret things. The sequence YLYEIAR, for example, is found almost exclusively in mammalian albumin, so if you're very sure you have that peptide you can be fairly sure you have albumin. If you report a protein that is significant with respect to your objectives or a hypothesis, a Western blot, if possible, would be important. You might want to check out this paper - ''What does it mean to identify a protein in proteomics?'': http://www.ncbi.nlm.nih.gov/pubmed/11852244.

It happens frequently. If you have a highly complex protein mixture (e.g. a cell lysate) your 2D dimension separation room is not big enough to separate all proteins into one single spot. You get overlayed spots and in mass spec you are able to identify more than one protein.

I'm surprised that you found more than one protein in only ONE of your spots! (Although you didn't mention the nature of your protein mixture.) As Thomas Halder suggests, with any form of chromatography, if you have two spots or two peaks you can be sure you have at least two analytes. A single peak or spot, however, may or may not represent a single substance. Even with high-efficiency gas or liquid chromatography, co-elution of one or more analytes is common. This is especially true with proteins on gels, which have relatively low resolving power. What's great about mass spectrometry is that the co-eluting substances can often be nicely analyzed based on molecular weight. 1D or 2D gels, preparative LC, or off-gel electrophoresis, are basically the first dimension of 2D separations, i.e., you will usually need to go at least one step further to completely understand your system.

Hi Masood,

we have recently published a paper that addresses the question you are asking in a lot of detail (essentially along the lines of the previous responses you have already received). Have a look at :

Thiede B, Koehler CJ, Strozynski M, Treumann A, Stein R, Zimny-Arndt U, Schmid M, Jungblut PR.:

High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-Orbitrap Mass spectrometer. Mol Cell Proteomics. 2013 Feb;12(2):529-38.

Hi Masood,

The assumption, that each spot on a 2D gel must be one protein is wrong. I agree with the other comments, 2DE does not have the resolution to separate proteins into one single spot. Generally, each spot contains a couple of proteins and of course the same protein can be detected from different spots due to post-translational modifications or proteolytic cleavage. The number of proteins you can detect from one spot depends on the method you use for identification, e.g. MALDI-TOF can detect less peptides than LC-MS/MS, so finally MALDI gives you less protein identified from one spot. Also important to know, that the concentration of the proteins in one spot is different. For example: in one spot there are three proteins A, B and C, where A protein has higher concentration than the other two. It means that using a less sensitive detection method you can miss the other two components and you assume wrongly that you have only one protein in the spot.

Of course, you can increase the resolution of your 2D gels by doing some pre-fractionataion, such as subcellular fractionation, chromatography-based fractionation, depletion of high-abundant proteins etc. If you want to do this, you should bear in mind that you need more samples, because you dilute your sample during pre-fractionation and of course you can loose some of proteins, too.

Thanks a lot @Thomas@John@Achim@Istvan

Can I report all proteins/peptides from one spot in article?

How do I justify the existence of multiple proteins in one spot?

Which characteristics of a protein is essential for reporting?

You SHOULD report all the proteins from a spot; as everyone here has noted, it's more-or-less expected that each spot probably represents more than one protein. With respect to what characteristics you should report: in general the more peptides with high-quality data (either exact masses or good CID spectra, the better. Three is probably a general minimum, but a single UNIQUE peptide with especially good data is sort-of OK if you don't over-interpret things. The sequence YLYEIAR, for example, is found almost exclusively in mammalian albumin, so if you're very sure you have that peptide you can be fairly sure you have albumin. If you report a protein that is significant with respect to your objectives or a hypothesis, a Western blot, if possible, would be important. You might want to check out this paper - ''What does it mean to identify a protein in proteomics?'': http://www.ncbi.nlm.nih.gov/pubmed/11852244.