Comprehensive online two-dimensional nanoLCxCZE-MS for deep top-down proteomics

Understanding proteoforms, i.e., the various molecular forms in which proteins can exist, is important for deciphering biological processes and diseases. While capillary zone electrophoresis (CZE) proved advantageous for proteoform separation, limited sample loading capabilities restrict its application. Here, we present a novel comprehensive two-dimensional nanoLCxCZE-MS platform for deep top-down proteomics (TDP). The 2D platform is highly automated, enabling robust performance and the possibility to perform proteoform quantitation as demonstrated by isobaric labeling experiments. The high orthogonality of reversed-phase LC and CZE leads to a peak capacity of 2200, leading to an increase in the number of identified proteoforms in a human Caucasian colon adenocarcinoma cell lysate sample by a factor of 3 compared to nanoLC-MS. Furthermore, CZE mobilities enable the attribution of many more proteoforms to a certain proteoform family on the MS1-level. Overall, the flexible platform enables highly efficient separation of intact proteoforms combined with sensitive MS-based TDP workflows, both for untargeted and targeted analysis of complex biological samples. Graphical AbstractWe report a robust and automated comprehensive nanoLCxCZE-MS platform for top-down proteomics. In addition to large volume sample injection and separation by hydrophobicity in the nanoLC, the orthogonal separation by CZE in the second dimension leads to a strong increase in peak capacity and, thus, in the number of identified proteoforms. CZE mobilities also enable the attribution of many more proteoforms to a proteoform family on the MS1-level. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/725123v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@df07b6org.highwire.dtl.DTLVardef@736d5corg.highwire.dtl.DTLVardef@10cef1org.highwire.dtl.DTLVardef@1825b55_HPS_FORMAT_FIGEXP M_FIG C_FIG