RESUMEN
The number of unique protein species in proteomes from a single mammalian cell type is not well defined but is likely to be at least 10000-20000. Since standard-size two-dimensional gels typically resolve only about 1500 to 3000 spots, they merely analyze a small portion of these proteomes. In addition, all insoluble proteins and typically proteins > 100 kDa are seldom resolved on two-dimensional (2-D) gels. The current study demonstrates the feasibility of an overall strategy for more comprehensive quantitative comparisons of complex proteomes derived from physiological fluids or mammalian cell extracts. A key feature of this approach is to prefractionate samples into a few well-resolved fractions based on the proteins' isoelectric points (pIs) using microscale solution isoelectric focusing. These fractions are then separated on narrow pH range two-dimensional gels approximately +/- 0.1 pH unit wider than the prefractionated pool. When this prefractionation approach is applied to complex mammalian proteomes, it improves resolution and spot recovery at high protein loads compared with use of parallel narrow pH range gels without prefractionation. The minimal cross-contamination between fractions allows quantitative comparisons in contrast to most alternative prefractionation methods. In addition, complementary data can be obtained by parallel analysis of the solubilized fraction on high-resolution large-pore-gradient one-dimensional gels followed by mass spectrometric identification to analyze proteins between 100 and approximately 500 kDa. Similarly, insoluble proteins can be analyzed using large-pore gels for large proteins and 10-12% one-dimensional sodium dodecyl sulfate (SDS) gels for smaller proteins. Together, these strategies should permit more reliable quantitative comparisons of complex mammalian proteomes where detection of at least 10000 protein spots is needed in order to analyze the majority of the unique protein species.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteoma/análisis , Animales , Concentración de Iones de Hidrógeno , MamíferosRESUMEN
Metabolically radiolabeled recombinant proteins were used to systematically evaluate peptide recoveries from in-gel trypsin digestion.At least 80% of the labeled tryptic peptides could be readily extracted from gel bands containing 1 to 10 pmol, and at least 70% could be extracted at 200- to 500-fmol levels using a recombinant 52-kd protein. Alkylation before electrophoresis or before trypsin digestion had minimal effects on peptide recovery; although alkylation, especially before gel analysis,may reduce heterogeneity of resulting peptides containing cysteines. Comparison of different gel thicknesses using unminced gel bands suggested that 1.0-mm gels were optimal. Surprisingly, peptide recoveries from 0.5-mm gels were low and variable, primarily because of increased diffusion of protein out of thin gels during fixing and staining. Although 70% to 85% of tryptic peptides could typically be extracted from gels over a range of conditions and protein concentrations, further processing of peptide extracts resulted in substantial additional losses. Even minimal handling resulted in loss of about 10% to 15% of extracted peptides by adsorption to plastic surfaces. Adsorptive losses were particularly high, sometimes exceeding 50%, and variable if extracts were partially dried in a Speedvac to concentrate the sample or to remove acetonitrile. High acetonitrile extraction and/or Speedvac concentration appear to be detrimental, and their elimination simplifies sample handling and automation. SYPRO Ruby Red, a sensitive noncovalent fluorescent stain appears to be an attractive alternative to Coomassie blue for in-gel trypsin digestion. These results suggest an optimized in-gel trypsin digestion strategy in which proteins in 1.0-mm-thick gels are stained with Coomassie blue or Ruby Red, digested overnight with modified trypsin, and extracted one or two times with small volumes of aqueous buffer. It is especially critical that subsequent surface exposure be minimized, and concentration by vacuum drying should be avoided.
RESUMEN
Four distinct spectrin alpha II domain polymorphisms are known to occur in several nonwhite populations. Type 1 is essentially the only form found in whites and is also the most common form in nonwhites. In contrast to most other spectrin mutations that are single-base substitutions, two of the alpha II domain polymorphisms, types 2 and 3, are particularly unusual because they appear to involve 4-Kd insertions relative to type 1. We have identified the mutations responsible for these polymorphisms using biochemical approaches and a computer database of spectrin-domain peptides separated by two-dimensional gels. The type 3 mutation is characterized by an apparent 4-Kd increase in alpha II domain peptides with no change in pI. This apparent molecular weight increase is a sodium dodecyl sulfate (SDS) gel artifact resulting from an Arg-->His mutation at residue 22 of the domain. The type 4 polymorphism shows a basic charge shift with no apparent change in molecular weight on gels. This charge shift results from a mutation of Thr-->Arg at position 174 of the domain. This mutation appears to be linked to a "silent" mutation at position 130 from an Ile-->Val. Support for possible linkage was obtained from analysis of three unrelated donors with the type 2 polymorphism. The type 2 polymorphism shows both the charge shift characteristic of the type 4 mutation and the apparent size shift that defines the type 3 polymorphism. Analysis of type 2 peptides confirmed that the two mutations described above for type 4 as well as the mutation at residue 22 observed in type 3 occur simultaneously in type 2. The observation that the type 2 polymorphism is a composite of the type 3 and 4 mutations is especially surprising because the type 2 polymorphism occurs far more frequently than either the type 3 or 4 forms. The basis for apparent linkage between the mutations at residues 130 and 174, which are encoded by different exons, is also not clear. Identification of the mutations described here permits design of genetic screening analyses that can be applied to larger populations to evaluate this potential linkage.
Asunto(s)
Espectrina/genética , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Mutación , Mapeo Peptídico , Polimorfismo Genético , Espectrina/químicaRESUMEN
Head-to-head association of two spectrin alpha beta heterodimers to form tetramers involves the formation of two equivalent alpha-beta complexes. The sites on the alpha subunit N-terminal region and beta subunit C-terminal region that form these alpha beta complexes have been identified using protease footprinting and direct binding assays. The existence of a similar previously hypothesized internal head-to-head alpha beta interaction in dimers was also demonstrated. The discrete regions of both subunits that are protected from proteolysis in tetramers and dimers are not due to the laterally associated subunit since head-to-head complexes of a univalent alpha peptide with a univalent beta peptide show similar protection of the same sites. These sites are unshielded immediately after monomers assemble side-to-side to form heterodimers, demonstrating that reconstituted dimers are initially in an "open" conformation. Conversion of open dimers to a closed form through formation of the internal head-to-head alpha beta association, as demonstrated by restoration of protease protection, occurred on a time scale of hours at 0 degrees C. Analysis of peptide binding affinities as well as isolation and sequence analysis of head-to-head alpha beta noncovalent complexes further defined the regions required for association on both subunits. These regions are homologous to the 106-residue repetitive motif that comprises most of both chains. An algorithm designed to improve prediction accuracy of multiple homologous motifs was used to model the conformation of spectrin repetitive motifs as well as the contact regions. In this model, the separate alpha and beta binding sites are incomplete complementary parts of a triple stranded folding unit. Formation of the alpha beta head-to-head complex produces a triple stranded conformational unit that is slightly different from other homologous motifs in the protein. Most hemolytic anemia mutations that are known to disrupt tetramer association are located in the mapped regions, including several mutations that induce a conformational change in the paired subunit.
Asunto(s)
Eritrocitos/metabolismo , Espectrina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Células Cultivadas , Humanos , Hidrólisis , Datos de Secuencia Molecular , Conformación Proteica , Espectrina/químicaRESUMEN
The mass-driven assembly of spectrin dimers to form tetramers involves two equal head-to-head alpha-beta associations and requires at least 30 degrees C for interconversion to occur readily. In this paper, the properties of tetramer formation were investigated using two complementary univalent peptides (the alpha I domain and beta monomers). Since the alpha I domain lacks an essential nucleation site required for side-to-side (lateral) heterodimer assembly [Speicher et al. (1992) J. Biol. Chem. 267, 14775-14782], these two peptides can only assemble head-to-head at a single site. This head-to-head assembly readily occurs at lower temperatures, indicating the temperature barrier for dimer-tetramer interconversion is caused by a conformational constraint of the dimer. This constraint, a closed hairpin loop, is released when the laterally associated partner is removed. The univalent alpha I-beta binding affinity at 37 degrees C (Ka = 1.4 x 10(5) M-1) is similar to the dimer-tetramer association constant at the same temperature. As the temperature is decreased from 37 to 0 degrees C, the alpha I-beta binding affinity increases about 32-fold. In contrast with head-to-head associations involving dimers, the second-order rate constants of two complementary univalent peptides (i.e., alpha I and beta) are dramatically higher, and the estimated activation energy (about 50 kJ mol-1) is about 5-fold lower. An open dimer conformation is an obligatory high-energy intermediate required for dimer-tetramer interconversion, and opening the dimer hairpin loop contributes about 190 kJ mol-1 to the activation energy for tetramer association.(ABSTRACT TRUNCATED AT 250 WORDS)