Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D2O, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while charge-stripping ions to lower charge values yields apparent folding as well as unfolding.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS (≈90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive. Here we use covalent chemical modification to reveal an attribute of spinal cord SOD1 common to both SOD1-linked FALS and SALS, but not present in normal or disease-affected tissues from other neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and spinal muscular atrophy, a non-ALS motor neuron disease. Biotinylation reveals a 32-kDa, covalently cross-linked SOD1-containing protein species produced not only in FALS caused by SOD1 mutation, but also in SALS. These studies use chemical modification as a novel tool for the detection of a disease-associated biomarker. Our results identify a shared molecular event involving a known target gene and suggest a common step in the pathogenesis between SALS and FALS.

Interleukin-17 (IL-17), the hallmark cytokine of T helper 17 (T_H17) cells, signals through a distinct receptor subclass, yet little is known about the mechanisms involved. IL-17 activates the expression of target genes through the actions of the transcription factors nuclear factor κB (NF-κB), CAAT enhancer binding protein δ (C/EBPδ), and C/EBPβ. The adaptor proteins tumor necrosis factor receptor–associated factor 6 (TRAF6) and Act1 are upstream of NF-κB and C/EBPδ, but the regulation of C/EBPβ remains undefined. Here, we show that IL-17 signaling led to phosphorylation of two sites in the regulatory 2 domain of C/EBPβ in a sequential, interdependent fashion. The first was rapid and dependent on extracellular signal–regulated kinase (ERK), whereas the second was dependent on the activity of glycogen synthase kinase 3β (GSK-3β). These pathways were mediated by distinct subdomains within IL-17 receptor A (IL-17RA). Whereas phosphorylation of threonine 188 (Thr¹⁸⁸) was mediated by the previously identified SEF/IL-17R homology domain–Toll-IL-1R–like loop (SEFIR-TILL), phosphorylation of Thr¹⁷⁹ occurred through a newly characterized motif located in the distal tail of IL-17RA. Phosphorylated C/EBPβ mediated a negative signal, because blocking ERK and GSK-3β increased expression of IL-17 target genes and a C/EBPβ-Thr¹⁸⁸ mutant enhanced activation of a C/EBP-dependent reporter. Overexpression of GSK-3β inhibited IL-17–induced activation of a C/EBP-dependent reporter, and Thr¹⁷⁹ of C/EBPβ was not phosphorylated in GSK-3β–deficient cells. Thus, IL-17 triggered the dual phosphorylation of C/EBPβ, which inhibited the expression of proinflammatory genes. This detailed dissection is the first for the IL-17–mediated C/EBP pathway and the first known example of a negative signal mediated by IL-17RA.