Coupling 2DLC with Online ESI-MS Detection

FIGURE 13.3 Raw and deconvoluted mass spectra of a yeast ribosomal protein (L16) from a 2DLC(SCX/RP)/MS experiment were obtained, where mass spectral adducts were observed because of insufficient washing of the second-dimension RP column (Panel a, 7 column volumes of wash). Panel b shows mass spectra for the same protein from an experiment with sufficient second-dimension wash volumes (Panel b, 14 column volumes of wash). 

FIGURE 13.4 Total ion chromatograms from the ID LC/MS analysis of a yeast ribosomal protein fraction separated using 0.1% TFA (Panel a) and 0.1% formic acid (Panel b) as mobile phase modifiers. TFA produced narrower, more concentrated, peaks for mass analysis that did not overcome the significant electrospray ionization suppression associated with using this modifier for LC/MS studies, resulting in an overall reduction in component intensities. 

The central engine of this data workflow is the process of spectral deconvolution. During spectral deconvolution, sets of multiply charged ions associated with particular proteins are reduced to a simplified spectrum representing the neutral mass forms of those proteins. Our laboratory makes use of a maximum entropy-based approach to spectral deconvolution (Ferrige et al., 1992a and b) that attempts to identify the most likely distribution of neutral masses that accounts for all data within the m/z mass spectrum. With this approach, quantitative peak intensity information is retained from the source spectrum, and meaningful intensity differences can be obtained by comparison of LC/MS runs acquired and processed under similar conditions. 

To process the LC/MS data more efficiently, we have automated this deconvolution functionality using a Visual Basic macro (termed AutoME or Automated Maximum 

FIGURE 13.5 The total ion chromatogram and deconvoluted protein mass map for a ID LC/MS analysis of yeast ribosomal proteins. The bubble size is proportional to component intensity. 

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