Fa Yun Che

Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brain and Pituitary

In neuropeptidomics, the degradation of a small fraction of abundant proteins overwhelms the low signals from neuropeptides, and many neuropeptides cannot be detected by mass spectrometry without extensive purification. Protein degradation was prevented when mice were sacrificed with focused microwave irradiation, permitting the detection of hypothalamic neuropeptides by mass spectrometry. Here we report an alternative and very simple method utilizing an ordinary microwave oven to inhibit enzymatic degradation. We used this technique to identify brain and pituitary neuropeptides. Quantitative analysis using mass spectrometry in combination with stable isotopic labeling was performed to determine the effect of microwave irradiation on relative levels of neuropeptides and protein degradation fragments. Microwave irradiation greatly reduced the levels of degradation fragments of proteins. In contrast, neuropeptide levels were increased about 2–3 times in hypothalamus by the microwave irradiation but not increased in pituitary. In a second experiment, three brain regions (hypothalamus, hippocampus, and striatum) from microwave-irradiated mice were analyzed. Altogether 41 neuropeptides or fragments of secretory pathway proteins were identified after microwave treatment; some of these are novel. These peptides were derived from 15 proteins: proopiomelanocortin, proSAAS, proenkephalin, preprotachykinins A and B, provasopressin, prooxytocin, melanin-concentrating hormone, proneurotensin, chromogranins A and B, secretogranin II, prohormone convertases 1 and 2, and peptidyl amidating monooxygenase. Although some protein degradation fragments were still found after microwave irradiation, these appear to result from protein breakdown during the extraction and not to an enzymatic reaction during the postmortem period. Two of the protein fragments corresponded to novel protein forms: VAP-33 with a 7-residue N-terminal extension and β tubulin with a glutathione on the Cys near the N terminus. In conclusion, microwave irradiation with an ordinary microwave oven effectively inhibits enzymatic postmortem protein degradation, increases the recovery of neuropeptides, and makes it possible to conduct neuropeptidomic studies with mouse brain tissues.

Identification of peptides from brain and pituitary of Cpefat/Cpefat mice

Cpefat/Cpefat mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpefat/Cpefat mice. These “orphan” peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzyme-substrate systems.

Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpefat/fat mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpefat/fat mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpefat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpefat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpefat/fat mice; these peptide processing intermediates with C‐terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.

Comprehensive Proteomic Analysis of Membrane Proteins in Toxoplasma gondii

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that is an important human and animal pathogen. Experimental information on T. gondii membrane proteins is limited, and the majority of gene predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T. gondii is important not only for understanding this parasites invasion mechanism(s), but also for the discovery of potential drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T. gondii, employing three proteomics strategies: one-dimensional gel liquid chromatography-tandem MS analysis (one-dimensional gel electrophoresis LC-MS/MS), biotin labeling in conjunction with one-dimensional gel LC-MS/MS analysis, and a novel strategy that combines three-layer “sandwich” gel electrophoresis with multidimensional protein identification technology. A total of 2241 T. gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially nonredundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T. gondii genome. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T. gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially suitable targets for the development of therapeutic strategies.

Nonprotein based enrichment method to analyze peptide cross-linking in protein complexes.

Cross-linking analysis of protein complexes and structures by tandem mass spectrometry (MS/MS) has advantages in speed, sensitivity, specificity, and the capability of handling complicated protein assemblies. However, detection and accurate assignment of the cross-linked peptides are often challenging due to their low abundance and complicated fragmentation behavior in collision-induced dissociation (CID). To simplify the MS analysis and improve the signal-to-noise ratio of the cross-linked peptides, we developed a novel peptide enrichment strategy that utilizes a cross-linker with a cryptic thiol group and using beads modified with a photocleavable cross-linker. The functional cross-linkers were designed to react with the primary amino groups in proteins. Human serum albumin was used as a model protein to detect intra- and intermolecular cross-linkages. Use of this protein-free selective retrieval method eliminates the contamination that can result from avidin-biotin based retrieval systems and simplifies data analysis. These features may make the method suitable to investigate protein-protein interactions in biological samples.

Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification

Phosphorylation is an important post-translational modification that rapidly mediates many cellular events. A key to understanding the dynamics of the phosphoproteome is localization of the modification site(s), primarily determined using LC-MS/MS. A major technical challenge to analysis is the formation of phosphopeptide-metal ion complexes during LC which hampers phosphopeptide detection. We have devised a strategy that enhances analysis of phosphopeptides, especially multiply phosphorylated peptides. It involves treatment of the LC system with EDTA and 2D-RP/RP-nanoUPLC-MS/MS (high pH/low pH) analysis. A standard triphosphorylated peptide that could not be detected with 1D-RP-nanoUPLC-MS/MS, even if the column was treated with EDTA-Na2 or if 25 mM EDTA-Na2 was added to the sample, was detectable at less than 100 fmol using EDTA-2D-RP/RP-nanoUPLC-MS/MS. Digests of α-casein and ß-casein were analyzed by EDTA-1D-RP-nanoUPLC, 2D-RP/RP-nanoUPLC, and EDTA-2D-RP/RP-nanoUPLC to compare their performance in phosphopeptide analysis. With the first two approaches, no tri- and tetraphosphopeptides were identified in either α- or ß-casein sample. With the EDTA-2D-RP/RP approach, 13 mono-, 6 di-, and 3 triphosphopeptides were identified in the α-casein sample, while 19 mono-, 8 di-, 4 tri-, and 3 tetraphosphopeptides were identified in the ß-casein sample. Using EDTA-2D-RP/RP-nanoUPLC-MS/MS to examine 500 μg of a human foreskin fibroblast cell lysate a total of 1,944 unique phosphopeptides from 1,087 unique phosphoproteins were identified, and 2,164 unique phosphorylation sites were confidently localized (Ascore ≥20). Of these sites 79% were mono-, 20% di-, and ∼1% were tri- and tetraphosphopeptides, and 78 novel phosphorylation sites in human proteins were identified.