High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus.

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.