Considerations of Concentration Dynamic Range

The enormous dynamic range of proteins in the sample represents an additional difficulty in proteome analysis. The best example is semm with a protein abundance ranging over eleven orders of magnitude (Anderson and Anderson, 2002). To detect the low abundant species, one has to load a sufficient amount of digest on a column to meet the limit of detection (LOD) of the MS instrument. Some reports published used up to 2.5 L of plasma with an extensive fractionation of intact proteins prior to LC-MS analysis on the peptide level (Rose et al., 2004). 

FIGURE 12.5 Human serum tryptic digest analysis. Fractionation in the first LC dimension was performed using a C18 column at pH 10. Fractions were analyzed using NanoEase 0.3 x 150 mm Atlantis d18 column. Approximately 66 lg (400 pmole of semm albumin peptides) was injected on column. Arrow points to a selected albumin peptide illustrating a local column mass overloading. Ten-5mm wide fractions were collected in 1st LC dimension. 

Depleting albumin and other major proteins from the sample combined with 2DLC-MS/MS analysis offer a promising solution (Bjorhall et al., 2005 Zolotaijova 

The consensus among proteome researchers is that separation is an essential part of complex protein analysis methods. A simplification of complex samples using 2DLC is beneficial for state-of-the-art MS/MS analysis. While 2DLC potentially provides a higher peak capacity than 1DLC, the orthogonality of separation has to be taken into consideration. 

It has been argued that in a typical 2DLC proteomic experiment, with only a limited number of fractions submitted for analysis in the second LC dimension, chromatographic peak capacity is less than 1000. This value is considerably lower than the expected sample complexity. Additional resolution is offered by MS, which represents another separation dimension. With the peak capacity defined as the number of MS/MS scans (peptide identifications) accomplished within the LC analysis time, the MS-derived peak capacity was estimated to be in an order of tens of thousands. While the MS peak capacity is virtually independent of LC separation performance, the complexity of the sample entering the MS instrument still defines the quality of MS/MS data acquisition. The primary goal of 2DLC separation is to reduce the complexity of the sample (and concentrate it, if possible) to a level acceptable for MS/MS analysis. What is the acceptable level of complexity to maintain the reliability and the repeatability of DDA experiments remains to be seen. 

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