Practical 2DLC Considerations in Proteome Research

As discussed above, in a typical proteomic experiment with a limited number of fractions collected, the practical peak capacity of 2DLC is well below 1000. This resolution is dramatically lower than the values considered in literature. 

The greater amount of peptide/protein identification in the RP-RP experiment is likely related to higher peptide recovery from the first LC dimension. In addition, a 

The proposed estimate has several limitations. When taking into account the limited orthogonality of investigated 2DLC modes, the practical peak capacity is reduced approximately to half. It needs to be also emphasized that a full separation power of the first LC dimension is realized only when the number of collected fractions exceeds its peak capacity (Murphy et al., 1998). If the number of fractions analyzed is low, the achievable chromatographic peak capacity suffers. 

A comparison of theoretical and practical peak capacity values, summarized in Table 12.2, leads to a conclusion that even the most promising 2DLC setups do not provide for the peak capacity needed to fully resolve a complex proteomic sample. As a result, the eluent entering the MS source typically contains multiple coeluting peptides. 

While chromatographic peak capacity is not adequate to resolve hundreds of thousands of components, many researchers argue that MS itself is an additional separation dimension with an orthogonal selectivity (separation is based on mass-to-charge ratio). Therefore, the combined resolution of LC and MS is greater than the chromatographically defined peak capacity. The question therefore stands What is the achievable peak capacity of the 2DLC-MS/MS system  

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