We describe the use of commercially available microcentrifugation devices (spin filters) for cleanup and digestion of protein samples for mass spectrometry analyses. The protein sample is added to the upper chamber of a spin filter with a > or = 3000 molecular weight cutoff membrane and then washed prior to resuspension in ammonium bicarbonate. The protein is then reduced, alkylated, and digested with trypsin in the upper chamber and the peptides are recovered by centrifugation through the membrane. The method provides digestion efficiencies comparable to standard in-solution digests, avoids lengthy dialysis steps, and allows rapid cleanup of samples containing salts, some detergents, and acidic or basic buffers.

The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl-N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in Vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in Vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in ViVo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.

Myoglobin (Mb) redox state affects meat color and is destabilized by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Our objective was to investigate lipid oxidation-induced oxymyoglobin (OxyMb) oxidation in Mb from two major meat-producing livestock species utilizing MS and proteomics tools. Porcine OxyMb was incubated with HNE and analyzed for metmyoglobin (MetMb) formation. MetMb formation was greater in the presence of HNE than controls at pH 7.4 and 37 degrees C (p <0.05). MALDI-TOF MS was used to identify adduct formation; only mono-adducts of HNE (via Michael addition) with porcine Mb were detected. LC-ESI-MS/MS identified three histidine (HIS) residues in porcine Mb that were readily adducted by HNE (HIS 24, 36 and 119), whereas in bovine Mb seven histidine residues (HIS 24, 36, 81, 88, 93, 119 and 152) were adducted. Quantitation of HNE-adducted peptides using isotope-labeled phenyl isocyanate indicated that, initially, HIS 36 was preferentially adducted in porcine Mb whereas HIS 81, 88 and 93 were the predominant sites of early HNE adduction in bovine Mb. Preferential HNE adduction at the proximal histidine (HIS 93) was observed exclusively in bovine OxyMb and may explain why lipid oxidation-induced OxyMb oxidation appears more extensive in beef, than in pork.

Human small ubiquitin-like modifier (sumo) proteins include sumo-1 and the less studied, nearly identical sumo-2 and sumo-3 proteins. Whereas the structurally related ubiquitin molecule targets proteins for degradation, sumo provides a distinct, yet poorly understood regulatory signal. Protein sumoylation is sensitive to diverse cellular stresses, yet the targets of sumoylation in stress are unknown. We studied protein sumoylation in HEK293 cells exposed to hydrogen peroxide, alkylating agents, and the lipid oxidation-derived electrophile 4-hydroxynonenal, which is an ubiquitous product of lipid oxidation associated with oxidative stress. Confocal immunofluorescence microscopy indicated that in unstressed cells sumo-1 targeted nuclear proteins, whereas sumo-2/3 targeted proteins in both nuclei and cytoplasm. Western blot analyses revealed changes in sumo-1 and sumo-2/3 targeting patterns with stress. We used immunoaffinity chromatography to harvest sumo-associated proteins from HA-sumo-1- and HA-sumo-3-expressing HEK293 cells both before and after treatment with 4-hydroxynonenal. Multidimensional liquid chromatography-tandem mass spectrometry analyses identified 54 HA-sumo-1-associated proteins and 38 HA-sumo-3-associated proteins. Major protein targets included RNA binding and processing proteins, transcription factors, metabolic enzymes, and cytoskeletal regulators. Treatment with 4-hydroxynonenal caused a near-complete redistribution of sumo-1 and sumo-3 to different protein targets, which included chaperones, antioxidant, and DNA damage signaling proteins. A 10-15% overlap of sumo-1 and sumo-3 targets before and after stress suggests that sumo proteins target distinct protein groups. The results suggest that reactive electrophiles not only directly modify proteins but also lead to indirect changes in endogenous protein modifications that regulate protein functions.