Efficient and clean charge derivatization of peptides for analysis by mass spectrometry.

Charge derivatization with succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) has great potential in several aspects of proteomics, such as peptide de novo sequencing, PTM analysis, etc. However, the excess reagent and its side products greatly limited its scope of use. Here, we report an improved method to perform charge derivatization of peptides by TMPP-Ac-OSu without interference from the excess reagent and corresponding side products. Briefly, the protein was first separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or coagulated with the gel. The protein in-gel was then incubated with a high concentration of reagent, followed by extensive washing. Afterwards, the protein was in-gel digested with trypsin according to the routine protocol. The mainly resultant peptides were attached with one positive tag on the N-termini or Lys-epsilon-NH(2). The process has been successfully applied to 2-DE resolved protein spots. Compared to the native proteins, the derivatized counterparts have higher rates of PMF identification and more straightforward tandem mass spectra. This promising method should pave the way for the practical use of charge derivatization in proteomics.

The hippocampal proteomic analysis of senescence-accelerated mouse: implications of Uchl3 and mitofilin in cognitive disorder and mitochondria dysfunction in SAMP8.

Senescence-accelerated mouse prone 8 (SAMP8) is considered as a useful animal model for age-related learning and memory impairments. Hippocampus, a critical brain region associated with cognitive decline during normal aging and various neurodegenerative diseases, appeared a series of abnormalities in SAMP8. To investigate the molecular mechanisms underlying age-related cognitive disorders, we used 2-DE coupled with MALDI TOF/TOF MS to analyze the differential protein expression of the hippocampus of SAMP8 at 6-month-old compared with the age-matched SAM/resistant 1 (SAMR1) which shows normal aging process. Two proteins were found to be markedly changed in SAMP8 as compared to SAMR1: ubiquitin carboxyl-terminal hydrolase L3 (Uchl3), implicating in cytosolic proteolysis of oxidatively damaged proteins, was down-regulated while mitofilin, a vital protein for normal mitochondria function, exhibited four isoforms with a consistent basic shift of isoelectric point among the soluble hippocampal proteins in SAMP8 compared with SAMR1. The alterations were confirmed by Western blotting analysis. The analysis of their expression changes may shed light on the mechanisms of learning and memory deficits and mitochondrial dysfunction as observed in SAMP8.

High-level expression of human TFF3 in Escherichia coli.

A strategy for expression and purification of recombinant N-terminal human trefoil factor family-domain peptide 3 (hTFF3) in Escherichia coli was established. The gene of hTFF3 was synthesized to substitute the low-usage condons with corresponding high-usage synonymous condons. At the same time, the signal peptide of DsbC was added to the N-terminus of the hTFF3 gene. The mature recombinant hTFF3 was located in the periplasm of E. coli, which can be released by sonication. The protein was further purified by a two-step cation exchange chromatography mentod. The yield is about 14-15 mg/l of culture. The biological activity of purified hTFF3 was analyzed by cell-based apoptosis assay, which shows that the recombinant hTFF3 is biologically active.

[Isolation and identification of proteins with anti-tumor and fibrinolysogen kinase activities from Eisenia foetida].

Proteins from Eisenia foetida possess many biological activities. A group of proteins precipitated by ethanol were isolated and purified by Sephadex G-75 and HiPrep 16/60 DEAE columns, then identified by one- or two- dimensional electrophoresis and mass spectrometry. 2D gel experiments displayed that the pI of proteins from Eisenia foetida were mainly from 3.0 to 4.0. Anti-tumor and kinase activities were determined by in vitro experiments. The enthanol fraction D2(8) showed both of the activities. These ethanol-precipitated proteins were identified further by native polyacrylamide electrophoresis, the protein spots were cut off from gels and digested by trypsin, the peptide mass fingerprints (PMFs) were determined by mass spectrometry. PMF, molecular weight, amino acid composition and N-terminus of 6 proteins were characterized, and band 9 was identified as D2(8). The results suggested that there exist proteins in Eisenia foetida possessed both anti-tumor and fibrinolysogen kinase activities. These methods can be used for identification of the natural bioactive proteins.

MilQuant: a free, generic software tool for isobaric tagging-based quantitation.

Isobaric tagging techniques such as iTRAQ and TMT are widely used in quantitative proteomics and especially useful for samples that demand in vitro labeling. Due to diversity in choices of MS acquisition approaches, identification algorithms, and relative abundance deduction strategies, researchers are faced with a plethora of possibilities when it comes to data analysis. However, the lack of generic and flexible software tool often makes it cumbersome for researchers to perform the analysis entirely as desired. In this paper, we present MilQuant, mzXML-based isobaric labeling quantitator, a pipeline of freely available programs that supports native acquisition files produced by all mass spectrometer types and collection approaches currently used in isobaric tagging based MS data collection. Moreover, aside from effective normalization and abundance ratio deduction algorithms, MilQuant exports various intermediate results along each step of the pipeline, making it easy for researchers to customize the analysis. The functionality of MilQuant was demonstrated by four distinct datasets from different laboratories. The compatibility and extendibility of MilQuant makes it a generic and flexible tool that can serve as a full solution to data analysis of isobaric tagging-based quantitation.

Comparison of two-dimensional gel electrophoresis based and shotgun strategies in the study of plasma membrane proteome.

Membrane proteins play important roles in various plasma membrane (PM) activities such as signal transduction and cell recognition. However, a comprehensive proteomic study of membrane proteins remains difficult. Different strategies have been employed to study PM proteome, but little effort has been made to systematically evaluate them. In the present work, liver PM was prepared by subcellular fractionation and an aliquot was washed by sodium carbonate. After evaluation of the PM fraction by electron microscopy and Western blotting, proteins in both original and carbonate washed PM were identified by either 2-DE coupled MALDI-TOF-MS or shotgun strategies. Then protein characteristics such as molecular weight, pI, grand average hydrophobicity, subcellular location, and transmembrane domains were systematically compared. The comparative analysis showed that shotgun strategies were more suitable to identify membrane proteins, while 2-DE-based strategies may serve as a complement. Furthermore, carbonate washing obviously enriched the integral membrane proteins. All the results suggested that the strategy combining carbonate washing and shotgun identification was the optimum strategy to study human liver PM proteome. Using this strategy, 260 high-confidence proteins were identified, wherein 139 were integral membrane proteins which had 1-17 transmembrane domains.

Proteomic analysis of rat pheochromocytoma PC12 cells.

PC12 cell line is well documented and widely applied as many kinds of models in neurobiological and neurochemical studies. Yet a thorough proteomic analysis has not been performed so far. Here we report the construction of a large-scale 2-D protein database for PC12 cells. The proteins extracted from PC12 cells were separated by 2-DE and identified by MALDI-TOF/TOF MS. A total of 1080 protein spots, excised from three different 2-D gels, were identified with high confidence. These proteins represent 474 different gene products, mainly binding proteins and enzymes. Three hundred and seven identified protein spots were located in the low-molecular weight region below 20 kDa. This database today represents one of the largest 2-D databases for higher eukaryotic cell proteomes and for low-molecular weight proteins. In addition, fragment ion spectra obtained by TOF/TOF confirmed that calcylin in PC12 cells was N-acetylated. The database of PC12 proteome is expected to be a powerful tool for neuroscientists.