Sheenah M. Mische

Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity

Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver‐stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver‐stained protein gel samples from one‐ or two‐dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.

Internal protein sequence analysis: Enzymatic digestion for less than 10 μg of protein bound to polyvinylidene difluoride or nitrocellulose membranes☆

A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.

Dorsal-Mediated Repression Requires the Formation of a Multiprotein Repression Complex at the Ventral Silencer

ABSTRACT Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila melanogaster embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknüllt(zen) contains Dorsal binding sites as well as an essential element termed AT2. We have identified and purified an AT2 DNA binding activity in embryos and shown it to consist of cut(ct) and dead ringer (dri) gene products. Studies of loss-of-function mutations in ct anddri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors.

Chemical Cleavage of Proteins in Solution

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins in solution. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS‐skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid‐proline (Asp‐Pro) peptide bonds; hydroxylamine cleaves at asparagine‐glycine (Asn‐Gly) peptide bonds, and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides.

Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-­1 kinase

Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1-313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14-313) by thrombin digestion during purification. The Pim-1 (14-313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 A resolution and belong to space group P6(5), with unit-cell parameters a = b = 95.9, c = 80.0 A, beta = 120 degrees and one molecule per asymmetric unit.

Amino Acid Analysis of PVDF Bound Proteins

Publisher Summary Amino acid analysis on polyvinylidene difluoride (PVDF) has been performed with high protein concentrations, but the accurate analyses of lower amounts have been more problematic. This chapter describes a reproducible manual hydrolysis method for determining the amino acid composition of a PVDF bound protein of one microgram. Five standard proteins ranging in molecular weight from 14.3 kD to 77 kD were electroblotted onto PVDF from SDS PAGE and then hydrolyzed, quantitatively eluted and analyzed by PTC amino acid analysis. The accuracy, precision, and recovery at 0.5, 1.0 and 2.0 micrograms were determined. An amino acid composition on 1 μg of electroblotted protein can be determined with 85% average accuracy, 5% average precision, and 88% average recovery. Lower levels (0.5 jug) can be determined with approximately 79% accuracy, 11% precision, and 32% average recovery.

Relation of streptococcal M protein with human and rabbit tropomyosin: The complete amino acid sequence of human cardiac alpha tropomyosin, a highly conserved contractile protein

Partial sequences of group A streptococcal M proteins exhibit up to 50% sequence identity with segments of rabbit skeletal tropomyosin. It is well recognized that rheumatic fever and rheumatic heart disease in humans are sequelae of group A streptococcal infection. To examine whether the human cardiac tropomyosin would exhibit greater homology with the streptococcal M proteins, we have now determined its complete amino acid sequence. The amino acid sequence of human cardiac tropomyosin was established from sequence analyses of its peptides derived by enzymic and chemical cleavages, and comparison of these sequences to the reported sequence of rabbit skeletal tropomyosin. These studies have revealed that the amino acid sequence of human cardiac alpha tropomyosin is identical to that of the rabbit skeletal alpha tropomyosin, but for a single conservative substitution of Arg/Lys at position 220. This observation increases the significance of the previously observed sequence homology between streptococcal M protein and rabbit skeletal tropomyosin and may have relevance to the pathogenesis of rheumatic fever. Furthermore, these results rank tropomyosin as one of the most highly conserved contractile proteins between vertebrate species reported thus far.

Enzymatic digestion of PVDF-bound proteins: A survey of sixteen non-ionic detergents

Publisher Summary This chapter describes the enzymatic digestion of polyvinylidene difluoride (PVDF)-bound proteins and presents a survey of sixteen non-ionic detergents. Successful enzymatic digestion of PVDF-bound protein requires the presence of non-ionic detergents in the digestion buffer. Direct mass analysis of peptide/detergent mixtures by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) or electrospray ionization mass spectrometry (ESI MS) cannot be obtained because of the presence of detergent related adducts signals and peptide signal suppression. As a result, peptide/detergent mixtures obtained from enzymatic digestion of PVDF-bound proteins cannot be used for quick screening and tentative identification of proteins via peptide mass database analysis search. Analysis of the primary structure of proteins is more powerful with the inclusion of MALDI-TOF MS data. The chapter describes (1) when high-performance liquid chromatography (HPLC) and Edman sequence analysis are to be employed; Genapol X-080, Pluronic F-127, and hydrogenated Triton X-100 produce a higher peptide recovery, while obtaining good sequencing initial yields, (2) when incorporating MALDI-TOF MS into the characterization scheme, the two glucopyranoside and two maltopyranoside detergents are good choices because of the strong signal response and absence of detergent clusters, (3) Tween 20 and Tween 80 are not recommended because of the lower sequencing initial yields and the detergents interference during MALDI-TOF MS analysis.

Assignment of cysteine and tryptophan residues during protein sequencing: Results of ABRF-94SEQ

Publisher Summary Routine assignment of cysteine and tryptophan residues is necessary for completeness of protein sequencing, because cysteine residue provides the major covalent cross-linkage in proteins and contributes to the biological and catalytic activity of many folded polypeptides. While, accurate assignment of tryptophan residues is crucial, when sequencing is used to design oligonucleotide probes of limited redundancy. This chapter discusses methods to improve the reliability of cysteine and tryptophan assignment, and describes the results of ABRF-94SEQ. ABRF-94SEQ allows to see which methods are used successfully in other facilities, and also provide the opportunity to assess these methods. It is observed that some who alkylate and some who does not alkylate, take risks making positive assignments for cysteine, when no PTH-residue or dehydroalanine/DTT adduct is apparent. If this assumption is done, it must be noted that this is not a positive assignment. Although cysteine and tryptophan identification may be problematic, the data suggest that routine identification of these residues should be possible for the majority of facilities.

A Synthetic Peptide for Evaluating Protein Sequencing Capabilities: Design of ABRF-93SEQ and Results

Publisher Summary This chapter describes a synthetic peptide for evaluating protein sequencing capabilities. In a study described in the chapter, the residue most frequently assigned correctly was V3 (77 positive correct and 0 tentative correct), followed closely by A4 and A10. C5 was the most difficult residue to call correctly and was the only residue with more tentative correct than positive correct assignments. It was also the residue most frequently assigned incorrectly and was most often mistaken for S (7/24), Y (6/24), or T (3/ 24). The difficulty with tryptophan assignment contrasted sharply with serine assignment, even though these were four of the first nine residues and the two serines were present as a doublet.