Direct analysis of protein complexes using mass spectrometry.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry.

Conventional capillary liquid chromatography/mass spectrometry (LC/MS) typically employs low microl/min flow rates with gas/liquid sheath to enhance spray stability. Over the past several years a number of reports have demonstrated success with electrospray (ES) interface designs optimized for submicroliter/min flows which have clear advantages in terms of enhancement of detection limit, lower sample consumption, and ability to accommodate a wider range of buffer conditions. We report here a fritless electrospray interface (FESI) design that is inexpensive and robust and can be operated and adapted to accommodate a variety of applications for submicroliter/min flow rates. The novelty of this interface revolves around the use of a fritless microcapillary column and precolumn application of electrospray voltage at a microtee junction to achieve stable microspray and nanospray flow rates. This sheathless FESI device eliminates postcolumn dead volume since small particles (

Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization.

We have developed a new system of chromosomal mutagenesis in order to study the functions of uncharacterized open reading frames (ORFs) in wild-type Escherichia coli. Because of the operon structure of this organism, traditional methods such as insertional mutagenesis run the risk of introducing polar effects on downstream genes or creating secondary mutations elsewhere in the genome. Our system uses crossover PCR to create in-frame, tagged deletions in chromosomal DNA. These deletions are placed in the E. coli chromosome by using plasmid pKO3, a gene replacement vector that contains a temperature-sensitive origin of replication and markers for positive and negative selection for chromosomal integration and excision. Using kanamycin resistance (Kn(r)) insertional alleles of the essential genes pepM and rpsB cloned into the replacement vector, we calibrated the system for the expected results when essential genes are deleted. Two poorly understood genes, hdeA and yjbJ, encoding highly abundant proteins were selected as targets for this approach. When the system was used to replace chromosomal hdeA with insertional alleles, we observed vastly different results that were dependent on the exact nature of the insertions. When a Kn(r) gene was inserted into hdeA at two different locations and orientations, both essential and nonessential phenotypes were seen. Using PCR-generated deletions, we were able to make in-frame deletion strains of both hdeA and yjbJ. The two genes proved to be nonessential in both rich and glucose-minimal media. In competition experiments using isogenic strains, the strain with the insertional allele of yjbJ showed growth rates different from those of the strain with the deletion allele of yjbJ. These results illustrate that in-frame, unmarked deletions are among the most reliable types of mutations available for wild-type E. coli. Because these strains are isogenic with the exception of their deleted ORFs, they may be used in competition with one another to reveal phenotypes not apparent when cultured singly.

Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12.

Mining the emerging abundance of microbial genome sequences for hypotheses is an exciting prospect of "functional genomics". At the forefront of this effort, we compared the predictions of the complete Escherichia coli genomic sequence with the observed gene products by assessing 381 proteins for their mature N-termini, in vivo abundances, isoelectric points, molecular masses, and cellular locations. Two-dimensional gel electrophoresis (2-DE) and Edman sequencing were combined to sequence Coomassie-stained 2-DE spots representing the abundant proteins of wild-type E. coli K-12 strains. Greater than 90% of the abundant proteins in the E. coli proteome lie in a small isoelectric point and molecular mass window of 4-7 and 10-100 kDa, respectively. We identified several highly abundant proteins, YjbJ, YjbP, YggX, HdeA, and AhpC, which would not have been predicted from the genomic sequence alone. Of the 223 uniquely identified loci, 60% of the encoded proteins are proteolytically processed. As previously reported, the initiator methionine was efficiently cleaved when the penultimate amino acid was serine or alanine. In contrast, when the penultimate amino acid was threonine, glycine, or proline, cleavage was variable, and valine did not signal cleavage. Although signal peptide cleavage sites tended to follow predicted rules, the length of the putative signal sequence was occassionally greater than the consensus. For proteins predicted to be in the cytoplasm or inner membrane, the N-terminal amino acids were highly constrained compared to proteins localized to the periplasm or outer membrane. Although cytoplasmic proteins follow the N-end rule for protein stability, proteins in the periplasm or outer membrane do not follow this rule; several have N-terminal amino acids predicted to destabilize the proteins. Surprisingly, 18% of the identified 2-DE spots represent isoforms in which protein products of the same gene have different observed pI and M(r), suggesting they are post-translationally processed. Although most of the predicted and observed values for isoelectric point and molecular mass show reasonable concordance, for several proteins the observed values significantly deviate from the expected values. Such discrepancies may represent either highly processed proteins or misinterpretations of the genomic sequence. Our data suggest that AhpC, CspC, and HdeA exist as covalent homomultimers, and that IcdA exists as at least three isoforms even under conditions in which covalent modification is not predicted. We enriched for proteins based on subcellular location and found several proteins in unexpected subcellular locations.

Identifying the major proteome components of Haemophilus influenzae type-strain NCTC 8143.

With the completion of the Haemophilus influenzae Rd genomic sequence, we know the identity of most of the theoretical proteins in the proteome of this bacterium. However, the most abundant components of the actual proteome are unknown. Using mass spectrometry and two-dimensional gel electrophoresis (2-DE), we sequenced and analyzed the most abundant proteins observed in the ATCC reference strain of H. influenzae, NCTC 8143 (303 of approximately 400 Coomassie-stained 2-DE spots). To automate the identification of 2-DE spots, we coupled a liquid autosampler to a microcolumn liquid chromatography electrospray ionization tandem mass spectrometer capable of identifying 22 spots per day. From the 303 sequenced spots, we identified 263 unique proteins. Most of the abundant proteins lie in an isoelectric point range of pH 4-7 and a molecular mass range of 10-100 kDa. Of the observed proteins, the most abundant is the outer membrane protein P2. Based on variety and abundance, proteins involved in energy metabolism and macromolecular synthesis are the dominant classes of proteins. Unexpectedly, tryptophanase was identified as a highly abundant protein in the strain NCTC 8143 whose sequence is not present in the genome of the Rd strain. By searching the tandem mass spectra against the translated genomic sequence, we identified several proteins which were not annotated in the genomic sequence. Surprisingly, 22% of the identified 2-DE spots represent isoforms in which gene products with the same primary sequence have different observed pI and M(r), indicating that these proteins are post-translationally processed. Although most proteins' predicted and observed isoelectric points and molecular masses show reasonable concordance, the observed values for several proteins deviate significantly from the predicted values. These anomalies may represent either highly processed proteins or misinterpretations of the genomic sequence. Using the technology developed in this project, the protein expression of other strains of H. influenzae grown under different environmental conditions can be compared to identify differences in their proteomes.

Future prospects for the analysis of complex biological systems using micro-column liquid chromatography-electrospray tandem mass spectrometry.

An overview is provided of methods for the study of complex biological processes by using micro-column liquid chromatography-electrospray ionization tandem mass spectrometry. Procedures discussed include electrospray ionization, micro-column liquid chromatography, tandem mass spectrometry, tandem mass spectra data interpretation for peptides, and database searching with mass spectral data. Several problems in immunology are discussed to illustrate this approach.

A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli.

A starvation-inducible DNA-binding protein was discovered as a result of the analysis of proteins synthesized in 3-day-old cultures of Escherichia coli. This 19-kD protein, designated Dps, is abundant in starved cells. In vitro, Dps forms extremely stable complexes with DNA, without apparent sequence specificity. When complexed with Dps, DNA is rendered DNase resistant. Mutant cells lacking Dps show dramatic changes in the pattern of proteins synthesized during starvation. The mutants also fail to develop starvation-induced resistance to hydrogen peroxide, an agent that can cause oxidative damage to DNA in vivo. These results have prompted us to postulate that Dps plays an important role both in gene expression and DNA protection during stationary phase. The existence of similar proteins, heretofore with no known function, in bacterial species distantly related to Escherichia coli suggests that Dps may define a novel class of widely conserved DNA-binding proteins.

Identification of proteins potentially involved in proximal-distal pattern formation in the regenerating forelimb of the newt.

We have used high resolution two-dimensional gel electrophoresis to identify and characterize proteins that may represent products of genes involved in establishing positional information along the proximal-distal axis of the regenerating forelimb of the newt Notophthalmus viridescens. At least 24 proteins have been found whose synthesis and (or) abundance is increased in proximal (midstylopodial) regenerates relative to midzeugopodial (distal) regenerates at either of two regeneration stages, the early dedifferentiation and moderate bud stages. Four of these same proteins show an axial asymmetry at both stages. Ten distal-specific proteins were also identified, although only one was common to both stages. More significantly, 6 of these 34 proteins (molecular masses of 73, 73, 51.5, 44.0, 19.5, and 16.5 kilodaltons and isoelectric points of 6.70, 6.74, 6.0, 6.05, 5.9, and 6.98, respectively) are regulated to proximal levels by treatment of distal regenerates with retinoic acid (RA) at both stages. An additional five are proximalized by RA at only one regeneration stage. Since the effect of RA is to proximalize positional information in blastema cells, these 11 proteins represent gene products that could be involved in a biochemical cascade leading to the establishment of positional information in the regenerating limb along this axis.

Drosophila genome project: one-hit coverage in yeast artificial chromosomes.

We present a strategy for assembling a physical map of the genome of Drosophila melanogaster based on yeast artificial chromosomes (YACs). In this paper we report 500 YACs containing inserts of Drosophila DNA averaging 200 kb that have been assigned positions on the physical map by means of in situ hybridization with salivary gland chromosomes. The cloned DNA fragments have randomly sheared ends (DY clones) or ends generated by partial digestion with either NotI (N clones) or EcoRI (E clones). Relative to the euchromatic portion of the genome, the size distribution and genomic positions of the clones reveal no significant bias in the completeness or randomness of genome coverage. The 500 mapped euchromatic clones contain an aggregate of approximately 100 million base pairs of DNA, which is approximately one genome equivalent of Drosophila euchromatin.

Integrated maps of the Drosophila genome: progress and prospects.

A physical map of the Drosophila melanogaster genome is being assembled, consisting of ordered overlapping cosmid clones. The map is constructed in steps, separately for each chromosomal division. Gaps in this map are to be bridged with yeast artificial chromosome clones. Hybridization to previously cloned genes and extensive use of in situ hybridization to polytene chromosomes ensure that the cosmid map is firmly anchored to the wealth of available genetic and cytogenetic information. The intention is to make the physical map widely available as part of an overall, integrated genetic resource for the Drosophila research community.

Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

He-T family DNA sequences in the Y chromosome of Drosophila melanogaster share homology with the X-linked stellate genes.

The genome of Drosophila melanogaster contains a class of repetitive DNA sequences called the He-T family, which is unusual in being confined to telomeric and heterochromatic regions. The specific He-T fragment designated Dm665 was cloned in yeast by selection for an autonomously replicating sequence (ARS). Dm665 contains a restriction fragment length polymorphism (RFLP) that is specific to males and thus derives from the Y chromosome. Deletion mapping using X-Y translocations indicates that sequences homologous to Dm665 occur in at least one major cluster in each arm of the Y chromosome. Among 20 yeast artificial chromosome (YAC) clones containing Drosophila sequences homologous with Dm665, four clones derive from defined regions of the long arm of the Y and two from the short arm. The sequence of Dm665 is 2443 bp long, consists of 59% A + T, and contains no significant open reading frames or direct or inverted repeats. However, Dm665 contains a region of 650 bp that shares homology with portions of the X-linked locus Stellate.

Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: improved methods, applications and gene cloning strategies.

The fields of protein chemistry and molecular biology are currently merging for study of biologically relevant events and conditions. To obtain partial sequences of microamounts of protein, efficient integration of high resolution separation and sequencing technologies is required. We report here on improved methods that allow extensive internal sequencing of 10 to 20 picomoles protein recovered from one- or two-dimensional gels. Each step of the standard protocol of Aebersold et al. (Proc. Natl. Acad. Sci. USA 1987, 84, 6970-6974) and the required instrumentation were examined and specifically adapted for use with submicrogram amounts of protein. Optimizations of in situ microdigests and liquid chromatography were needed for improved peptide recovery. Subsequent automated sequencing required subpicomole analysis. New methods for S-alkylation of gel-separated proteins and accurate identification of tryptophan-containing peptides were introduced to insure overall higher efficiencies. The acquired internal sequences facilitated cloning of the genes and several strategies are discussed. Applying our method, several proteins of unknown structure were sequenced and successfully identified or cloned. Internal sequences of submicrogram protein amounts, recovered from a single two-dimensional gel of Escherichia coli total protein (120 micrograms), allowed unambiguous identification of the spots but pre-gel enrichment will be required for analysis of most (90-95%) other spots. Integration of comprehensive two-dimensional gel protein databases with methods and strategies outlined here could potentially be an abundant source of DNA probes and markers useful for guidance of the human genome sequencing project and for analysis of the emerging vast amounts of data.