Identification of proteins by mass spectrometry (MS) is an essential step in proteomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.
Algorithms ; Peptide Mapping ; methods ; statistics & numerical data ; Proteomics ; methods ; statistics & numerical data ; Tandem Mass Spectrometry

The aim of this study was to explore the distinct protein profiles of different subtype of acute myeloid leukemia (AML), including M(1), M(2), M(3) and acute lymphoid leukemia (ALL) by differential proteomic expression analysis. The proteins of bone marrow leukemia cells from AML and ALL patients were separated by two-dimensional electrophoresis (2-DE). 2-DE patterns were analyzed by PDQuest 7.4 software and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and bioinformatics. The results indicated that 21 differentially expressed proteins were found by 2-DE and 15 were identified by MS to be significantly differentially expressed. In AML, seven proteins were highly expressed such as MPO, PRDX3, CALR and ECH1 and so on, and eight proteins were highly expressed in ALL, including ARHGDIB, PFN1 and ACTG1 and so on. It is concluded that the distinct protein profiles between AML and ALL have been proved. It may be helpful for the identification of new targets for specific treatment approaches and the molecular markers for the early diagnosis of leukemia.
Humans ; Leukemia, Myeloid, Acute ; metabolism ; Peptide Mapping ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; Proteome ; Proteomics

Biomarkers ; Humans ; Male ; Peptide Mapping ; Prognosis ; Prostatic Neoplasms ; blood ; Proteome ; metabolism

With the rapid development of MS technologiesy, the demands for a more sophisticated MS interpretation algorithm haves grown as well. We have developed a new protein fingerprinting method using a binomial distribution, (fBIND). With the fBIND, we improved the performance accuracy of protein fingerprinting up to the maximum 49% (more than MOWSE) and 2% than(at a previous binomial distribution approach studied by of Wool et al.) as compared to the established algorithms. Moreover, we also suggest a the statistical approach to define the significance of transcription factors and motifs in the identified proteins based on the Gene Ontology (GO).
Binomial Distribution ; Fungal Proteins* ; Gene Ontology ; Peptide Mapping ; Transcription Factors* ; Wool ; Yeasts*

OBJECTIVE: To establish 2-dimensional polyacrylamide gel electrophoresis (2-DE) map from human nasal polyps and normal nasal mucosa, and to identify differential expression proteins of 2-DE map. METHODS: Samples of nasal polyps and nasal mucosa (each sample group containing 7 cases) were obtained. The total proteins were extracted and separated by immobilized pH gradient (IPG)-based 2-DE. The silver-stained 2-DE was scanned with digital Imagescanner and analyzed with ImageMaster 2-DE Elite 4.01 software. To obtain peptide mass fingerprint (PMF) of differential protein spots, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used. The PMF was searched in Swiss-Prot and TreMBL database by Pept-Ident software, to identify differential expression proteins. RESULTS: The well-resolved, reproducible 2-DE maps of nasal polyps and nasal mucosa were established. For the polyps tissues, the average proteins spot of three 2-DE maps was 825+/-78; and 682+/-96 spot was matched with the average matching rate of 82.7%. The average deviations of matched spot position were (1.13+/-0.16) mm in IEF direction and (1.45+/-0.21) mm in SDS-PAGE direction, respectively. For the nasal mucosa tissues, the average proteins spot of three 2-DE maps was 936+/-62; and 821+/-78 spots were matched with the average matching rate of 87.7%. After comparing the 2-DE maps of nasal polyps and nasal mucosa tissues, the protein spots were 1,458 and 1,617 respectively; and 1,026 protein spots were matched. Forty differential expression protein spots were incised from silver staining gel randomly and digested in the gel by TPCK-Trypsin. Thirty-four PMFs were obtained by MALDI-TOF-MS and 24 differential proteins were identified. CONCLUSION The well-resolved, reproducible 2-DE maps of human nasal polyps and nasal mucosa have been successfully established. Certain differential proteins related to the pathogenesis of human nasal polyps are identified.
Adult ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Male ; Middle Aged ; Nasal Polyps ; metabolism ; Peptide Mapping ; Proteomics

OBJECTIVETo evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis (2-DE)-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein.

METHODSDirect solubilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry.

RESULTSThe total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675-/+46 and 702-/+49, respectively. The average matching protein spots were 573-/+42 and 623-/+52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85-/+0.30 mm and 0.81-/+0.28 mm in IEF direction, and 1.02-/+0.18 mm and 0.97-/+0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein.

CONCLUSIONHot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.

Blood Protein Electrophoresis ; Blood Proteins ; analysis ; isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Peptide Mapping ; Protein Interaction Mapping ; Reproducibility of Results

This study was purposed to find new biomarkers and to establish protein finger print model for diagnosis of leukemia. A total of 40 leukemia samples and 37 healthy control samptes were tested by surface enhance laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF- MS). The data of spectra were analyzed by bioinformatics tools like Biomarker Patterns 5.0 and discriminant analysis to establish diagnostic mode1. The results showed that 22 protein features were stably detected by protein fingerprint, The detective model combined with 3 biomarkers (m/z 4650, 8609 and 11660) could differentiate leukemia with sensitivity of 97.5% (39/40) and specificity of 91.9%(34/37). It is concluded that the detective model established by 3 protein features may be a novel method for diagnosis of leukemia.
Biomarkers, Tumor ; analysis ; Humans ; Leukemia ; diagnosis ; Molecular Weight ; Peptide Mapping ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

OBJECTIVETo set up the dual-indexes sequence analytical method for far-infrared fingerprint in which the dual indexes are common peak ratio and variant ration.

METHODTwo new indexes, common peak ratio and variant peak ratio, were applied and their values were calculated by means of sequential analysis, in which each Pyritum sample's far-infrared fingerprint spectra were set up and the common peak ratio sequences were arranged in order of size in comparision with other samples.

RESULTThe analytical results suggested that samples S3 and S4, S5, S6 and S7, S8 and S9 from the same region showed higher common peak ratio and lower variant peak ratio. However, the sample S1 from Anhui showed little similarity with others.

CONCLUSIONThe method, applied to distinguish Pyritum of different areas and batches, is reasonable to characterize of traditional Chinese medicine.

Iron ; analysis ; chemistry ; Medicine, Chinese Traditional ; Peptide Mapping ; Quality Control ; Sequence Analysis, Protein ; Spectrophotometry, Infrared ; methods ; Sulfides ; analysis ; chemistry

OBJECTIVETo compare the proteomic differences between normal and necrozoospermic human spermatozoa.

METHODSWe isolated proteins from 30 ejaculates of 10 healthy donors and 3 specimens of a necrozoospermia patient by two-dimensional gel electrophoresis (2-DE), analyzed their 2-DE maps and identified some differentially expressed proteins by mass spectrometry.

RESULTSA total of (905 +/- 57) and (792 +/- 28) proteins were isolated from the spermatozoal maps of the normal and necrozoospermic men, respectively, and 178 found to be differentially expressed in that of the necrozoospermia patient. The 6 missing protein spots in the necrozoospermic map were identified as sperm protein (human, accession No. 060904), zinc finger protein 174 (AW-1), F-actin capping protein alpha-3 subunit, testis-specific inhibitor of apoptosis, death domain receptor 3 soluble form (fragment) and peptide similar to the activator of CREM in the testis.

CONCLUSIONSix missing protein spots were identified in the necrozoospermic spermatozoal map which may be associated with the development of necrozoospermia.

Electrophoresis, Gel, Two-Dimensional ; Humans ; Male ; Mass Spectrometry ; Oligospermia ; metabolism ; pathology ; Peptide Mapping ; Proteome ; analysis ; Proteomics ; methods ; Spermatozoa ; metabolism ; pathology

The application of in vitro selection method to isolate nucleic acids, peptides and proteins according to their functions has been studied intensively in recent years. In vitro display technologies are not limited by cellular transformation efficiencies; thus, very large libraries of up to 10(13)-10(14) members can be built. The most popular in vitro display technologies are ribosome display and mRNA display; ribosome display achieves the mRNA-ribosome-nascent peptide complexes by stalling the translating ribosome in an in vitro translation reaction. In mRNA display, the mRNA-protein complex is achieved by binding the two macromolecules through a small adaptor molecule, typically puromycin; these mRNA-peptide fusions can then be purified and subjected to in vitro selection. In vitro display technologies provide a different approach to the in vitro selection and directed evolution of peptides and proteins. This review focuses on the principle and method of ribosome display and mRNA display technologies, and discusses their applications.
Animals ; Directed Molecular Evolution ; Gene Library ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; RNA, Messenger ; chemistry ; genetics ; Ribosomes ; chemistry ; genetics