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Disulfide bonds in proteins have a substantial impact on protein structure, stability, and biological activity. Localizing disulfide bonds is critical for understanding protein folding and higher-order structure. Conventional top-down mass spectrometry (TD-MS), where only terminal fragments are assigned for disulfide-intact proteins, can access disulfide information, but suffers from low fragmentation efficiency, thereby limiting sequence coverage. Here, we show that assigning internal fragments generated from TD-MS enhances the sequence coverage of disulfide-intact proteins by 20-60% by returning information from the interior of the protein sequence, which cannot be obtained by terminal fragments alone. The inclusion of internal fragments can extend the sequence information of disulfide-intact proteins to near complete sequence coverage. Importantly, the enhanced sequence information that arise from the assignment of internal fragments can be used to determine the relative position of disulfide bonds and the exact disulfide connectivity between cysteines. The data presented here demonstrates the benefits of incorporating internal fragment analysis into the TD-MS workflow for analyzing disulfide-intact proteins, which would be valuable for characterizing biotherapeutic proteins such as monoclonal antibodies and antibody-drug conjugates.

Martin GG, Atshaves BP, Huang H, McIntosh AL, Williams B J, Pai P, Russell DH, Kier AB, Schroeder F. Hepatic phenotype of liver fatty acid binding protein (L-FABP) gene-ablated mice. Am J Physiol Gastrointest Liver Physiol 297: G1053-G1065, 2009. First published October 8, 2009; doi: 10.1152/ajpgi.00116.2009.--Although the function of liver fatty acid binding protein in hepatic fatty acid metabolism has been extensively studied, its potential role in hepatic cholesterol homeostasis is less clear. Although hepatic cholesterol accumulation was initially reported in L-FABP-null female mice, that study was performed with early N2 backcross generation mice. To resolve whether the hepatic cholesterol phenotype in these [L-FABP.sup.-/-] mice was attributable to genetic inhomogeneity, these [L-FABP.sup.-/-] mice were further backcrossed to C57B1/6 mice up to the N10 (99.9% homogeneity) generation. Hepatic total cholesterol accumulation was observed in female, but not male, [L-FABP.sup.-/-] mice at all (N2, N4, N6, N10) backcross generations examined. The greater total cholesterol was due to increased hepatic levels of both unesterified (free) cholesterol and esterified cholesterol. Altered hepatic cholesterol accumulation correlated directly with L-FABP's ability to bind cholesterol with high affinity as shown by direct L-FABP binding of fluorescent cholesterol analogs (NBD-cholesterol, dansyl- cholesterol), a photoactivatable cholesterol analog [free cholesterol benzophenone (FCBP)], and free cholesterol (circular dichroism, isothermal titration microcalorimetry). One mole of fluorescent sterol was bound per mole of L-FABP. This was confirmed by photo-cross-linking studies with the photoactivatable cholesterol analog FCBP and by isothermal titration calorimetry with free cholesterol, which showed that L-FABP bound only one sterol molecule per L-FABP molecule. In contrast, the hepatic phenotype of male, but not female, [L-FABP.sup.-/-] mice was characterized by decreased hepatic triacylglycerol levels at all backcross generations examined. Taken together, these data support the hypothesis that L-FABP plays a role in physiological regulation of not only hepatic fatty acid metabolism, but also that of hepatic cholesterol. mouse; gene ablation; triacylglycerol doi: 10.1152/ajpgi.00116.2009

A new method for displaying CE-MALDI-MS data for proteolytic digests is described. This data display mode yields distinct charge-based trends for plots of mass-to-charge (m/z) versus CE migration time. These trends arise owing to the in-solution charge state of the peptides, and this interpretation was confirmed by using empirical peptide electromigration models and peptide standards as charge-state markers. These charge-state specific trends exhibit analytical utility by providing additional chemical information about the peptides, which increases the confidence level of peptide identification and provides a rapid means for screening for posttranslationally modified peptides.