High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification.

Identification of anonymous proteins from two-dimensional (2-D) gels by peptide mass fingerprinting is one area of proteomics that can greatly benefit from a simple, automated workflow to minimize sample contamination and facilitate high-throughput sample processing. In this investigation we outline a workflow employing robotic automation at each step subsequent to 2-D gel electrophoresis. As proof-of-concept, 96 protein spots from a 2-D gel were analyzed using this approach. Whole protein (1 mg) from mature, dry soybean (Glycine max [L.] Merr.) cv. Jefferson seed was resolved by high resolution 2-D gel electrophoresis. Approximately 150 proteins were observed after staining with Coomassie Blue. The rather low number of detected proteins was due to the fact that the dynamic range of protein expression was greater than 100-fold. The most abundant proteins were seed storage proteins which in total represented over 60% of soybean seed protein. Using peptide mass fingerprinting 44 protein spots were identified. Identification of soybean proteins was greatly aided by the use of annotated, contiguous Expressed Sequence Tag (EST) databases which are available for public access (UniGene, ftp.ncbi.nih.gov/repository/UniGene/). Searches were orders of magnitude faster when compared to searches of unannotated EST databases and resulted in a higher frequency of valid, high-scoring matches. Some abundant, non seed storage proteins identified in this investigation include an isoelectric series of sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins. This survey of anonymous seed proteins will serve as the basis for future comparative analysis of seed-filling in soybean as well as comparisons with other soybean varieties.

[1]  Liangjiang Wang,et al.  Mapping the Proteome of Barrel Medic (Medicago truncatula)1,212 , 2003, Plant Physiology.

[2]  M. Schuler,et al.  Closely related families of genes code for the α and α‘ subunits of the soybean 7S storage protein complex , 1982 .

[3]  S. Barker,et al.  Soybean beta-conglycinin genes are clustered in several DNA regions and are regulated by transcriptional and posttranscriptional processes. , 1989, The Plant cell.

[4]  L. Wagner,et al.  21. UniGene: A Unified View of the Transcriptome , 2003 .

[5]  B. Larkins,et al.  Protein Storage Bodies and Vacuoles , 1999, Plant Cell.

[6]  A. Esen,et al.  Heterogeneity of soybean seed proteins: one-dimensional electrophoretic profiles of six different solubility fractions. , 1981, Journal of agricultural and food chemistry.

[7]  H. Peng,et al.  Mutations affecting induction of glycolytic and fermentative genes during germination and environmental stresses in Arabidopsis. , 1999, Plant physiology.

[8]  J. Cottrell,et al.  Protein identification by peptide mass fingerprinting. , 1994, Peptide research.

[9]  J. T. Madison,et al.  The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits. , 1982, The Journal of biological chemistry.

[10]  M. Schuler,et al.  Structural sequences are conserved in the genes coding for the α, α′ and β-subunits of the soybean 7S seed storage protein , 1982 .

[11]  A. Esen,et al.  Heterogeneity of soybean proteins: two-dimensional electrophoretic maps of three solubility fractions , 1982 .

[12]  P. Overvoorde,et al.  Topographical analysis of the plasma membrane-associated sucrose binding protein from soybean. , 1994, The Journal of biological chemistry.

[13]  A. Kinney,et al.  Genetic Modification Removes an Immunodominant Allergen from Soybean1,212 , 2003, Plant Physiology.

[14]  M. Loureiro,et al.  The soybean sucrose binding protein gene family: genomic organization, gene copy number and tissue-specific expression of the SBP2 promoter. , 2003, Journal of experimental botany.

[15]  Cottrell Js,et al.  Protein identification by peptide mass fingerprinting. , 1994 .

[16]  H. Rolletschek,et al.  Energy Status and Its Control on Embryogenesis of Legumes. Embryo Photosynthesis Contributes to Oxygen Supply and Is Coupled to Biosynthetic Fluxes1 , 2003, Plant Physiology.

[17]  N. Anderson,et al.  Multi‐component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome , 2003, Proteomics.

[18]  D. Hochstrasser,et al.  Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. , 1996, Biotechnology & genetic engineering reviews.

[19]  R. Scopes,et al.  Analysis of Proteins , 1998 .

[20]  Daniel C. Liebler.  Protein Identification by Peptide Mass Fingerprinting , 2002 .

[21]  Kent Vander Velden,et al.  The proteome of maize leaves: Use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints , 2001, Electrophoresis.

[22]  R. Krishna,et al.  Post-translational modification of proteins. , 1993, Advances in enzymology and related areas of molecular biology.

[23]  G. Ditta,et al.  Developmental regulation of cloned superabundant embryo mRNAs in soybean. , 1981, Developmental biology.

[24]  H. Hauser,et al.  The structure, physical and chemical properties of the soy bean protein glycinin. , 1975, Biochimica et biophysica acta.

[25]  K. Shibasaki,et al.  Heterogeneity of beta-conglycinin. , 1976, Biochimica et biophysica acta.

[26]  M. Mann,et al.  Analysis of proteins and proteomes by mass spectrometry. , 2001, Annual review of biochemistry.

[27]  K. Shibasaki,et al.  Major proteins of soybean seeds. A straightforward fractionation and their characterization. , 1976, Journal of agricultural and food chemistry.

[28]  V. Franceschi,et al.  A 62-kD sucrose binding protein is expressed and localized in tissues actively engaged in sucrose transport. , 1992, The Plant cell.

[29]  B. Scallon,et al.  Characterization of the glycinin gene family in soybean. , 1989, The Plant cell.

[30]  M. Mann,et al.  Proteomics to study genes and genomes , 2000, Nature.

[31]  C. Rameau,et al.  Peas: Genetics, Molecular Biology and Biotechnology , 1993 .

[32]  W. Hurkman,et al.  Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. , 1986, Plant physiology.

[33]  W. Chao,et al.  A Plasma Membrane Sucrose-binding Protein That Mediates Sucrose Uptake Shares Structural and Sequence Similarity with Seed Storage Proteins but Remains Functionally Distinct* , 1997, The Journal of Biological Chemistry.

[34]  M R Wilkins,et al.  Strategy for protein isoform identification from expressed sequence tags and its application to peptide mass fingerprinting , 2001, Proteomics.

[35]  H. Hirano,et al.  Nucleotide Sequence of the Basic 7S Globulin Gene from Soybean , 1994, Plant physiology.

[36]  Wayne F. Patton,et al.  Ultrasensitive fluorescence protein detection in isoelectric focusing gels using a ruthenium metal chelate stain , 2000, Electrophoresis.

[37]  T. Hunkapiller,et al.  Peptide mass maps: a highly informative approach to protein identification. , 1993, Analytical biochemistry.

[38]  N. Tumer,et al.  Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors. , 1981, The Journal of biological chemistry.

[39]  C. Kannicht,et al.  2D-electrophoresis. Detection of glycosylation and influence on spot pattern. , 2002, Methods in molecular biology.

[40]  R. Kennedy,et al.  Genetic and biochemical analysis of anaerobically-induced enzymes during seed germination of Echinochloa crus-galli varieties tolerant and intolerant of anoxia. , 2003, Journal of experimental botany.

[41]  K. Shibasaki,et al.  Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms. , 1977, Biochimica et biophysica acta.

[42]  H. Hirano,et al.  Soybean basic 7 S globulin represents a protein widely distributed in legume species , 1987 .