Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures

Mirgorodskaya, Ekaterina; Braeuer, Corina; Fucini, Paola; Lehrach, Hans; Gobom, Johan

doi:10.1002/pmic.200400984

Cited by 79 publications

(71 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The well established on-line coupling of nLC to an ESI instrument is more straightforward and thus better understood in regard to its problems than the MALDI spotting procedure (52). The loss of separation power through the MALDI spotting process can hardly be compensated despite the advantage of the off-line measurement.…”

Section: Discussionmentioning

confidence: 99%

Elastase Digests

Rietschel

Arrey

Meyer

et al. 2009

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.

show abstract

Section: Discussionmentioning

confidence: 99%

Elastase Digests

Rietschel

Arrey

Meyer

et al. 2009

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…(iv) Nano-LC-MALDI MS. Two samples for which reliable protein identification could not be achieved by direct MALDI MS analysis of the tryptic digests were analyzed by nano-liquid chromatography (LC)-MALDI MS as described previously (23). In brief, peptide samples were loaded on an 1100 Series model nanoflow LC system (Agilent Technologies, Waldbronn, Germany).…”

Section: Methodsmentioning

confidence: 99%

Identification of a Chlorobenzene Reductive Dehalogenase in Dehalococcoides sp. Strain CBDB1

Adrian

Rahnenführer

Gobom

et al. 2007

Appl Environ Microbiol

Self Cite

128

114

View full text Add to dashboard Cite

A chlorobenzene reductive dehalogenase of the anaerobic dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 was identified. Due to poor biomass yields, standard protein isolation procedures were not applicable. Therefore, cell extracts from cultures grown on trichlorobenzenes were separated by native polyacrylamide gel electrophoresis and analyzed directly for chlorobenzene reductive dehalogenase activity within gel fragments. Activity was found in a single band, even though electrophoretic separation was performed under aerobic conditions. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and nano-liquid chromatography-MALDI MS analysis of silver-stained replicas of the active band on native polyacrylamide gels identified a protein product of the cbdbA84 gene, now called cbrA. The cbdbA84 gene is one of 32 reductive dehalogenase homologous genes present in the genome of strain CBDB1. The chlorobenzene reductive dehalogenase identified in our study represents a member of the family of corrinoid/iron-sulfur cluster-containing reductive dehalogenases. No orthologs of cbdbA84 were found in the completely sequenced genomes of Dehalococcoides sp. strains 195 and BAV1 nor among the genes amplified from Dehalococcoides sp. strain FL2 or mixed cultures containing Dehalococcoides. Another dehalogenase homologue (cbdbA80) was expressed in cultures that contained 1,2,4-trichlorobenzene, but its role is unclear. Other highly expressed proteins identified with our approach included the major subunit of a protein annotated as formate dehydrogenase, transporter subunits, and a putative S-layer protein.Dehalococcoides sp. strain CBDB1 belongs to a phylogenetically isolated cluster of strictly anaerobic bacteria that use chlorinated compounds in their energy metabolism by coupling reductive dehalogenation to electron transport phosphorylation (2, 22). Strain CBDB1 uses polychlorinated benzenes, phenols, and dibenzodioxins as growth-supporting electron acceptors (2, 3, 5, 13). Among the chlorobenzenes, 1,2,3-trichlorobenzene (TCB), 1,2,4-TCB, all tetrachlorobenzene (TeCB) isomers, and penta-and hexachlorobenzene are dechlorinated (2, 10, 13). Reductive dechlorination of chlorinated benzenes was also demonstrated for the Dehalococcoides ethenogenes strain 195 (7), a strain that was originally cultivated with chlorinated ethenes as electron acceptors (22), and for Dehalococcoides-like bacterium DF-1, identified in a mixed culture (38).Several enzymes catalyzing the respiratory reductive dechlorination of chloroaromatics have been isolated and characterized, e.g., the chlorophenol dehalogenases of Desulfitobacterium spp. (6, 15, 36) and the 3-chlorobenzoate dehalogenase of Desulfomonile tiedjei (30). Attempts to isolate chlorobenzene reductive dehalogenase from Dehalococcoides sp. strain CBDB1 have been hampered by poor biomass yields (10). However, the characterization of chlorobenzene reductive dehalogenase activity in cell extracts of strain CBDB1 showed that this enzyme shared several properties wit...

show abstract

“…However, standardization and automation of two-dimensional gel electrophoresis has proved difficult and the use of the resulting protein patterns as proteomic reference maps has only been successful in a few cases. A complementary technique, liquid chromatography, is easier to automate, and it can be directly coupled to mass spectrometry 63,64 . Minimized surface areas of nanoliquid chromatographical systems reduce analyte losses due to surface adsorption and dead volumes (the amount of overfill left in the tubing after dispensing is complete).…”

Section: Post-translational Modifications and Protein Profilesmentioning

confidence: 99%

Miniaturization in functional genomics and proteomics

et al. 2005

Self Cite

View full text Add to dashboard Cite

| Proteins are the key components of the cellular machinery responsible for processing changes that are ordered by genomic information. Analysis of most human proteins and nucleic acids is important in order to decode the complex networks that are likely to underlie many common diseases. Significant improvements in current technology are also required to dissect the regulatory processes in high-throughtput and with low cost. Miniaturization of biological assays is an important prerequisite to achieve these goals in the near future. SYSTEMS BIOLOGYThe study of the complex interactions that occur at all levels of biological information -from whole-genome sequence interactions to developmental and biochemical networks -and their functional relationship to organism-level phenotypes. NATURE REVIEWS | GENETICS VOLUME 6 | JUNE 2005 | 465 and concomitantly reducing the complexity of genomic DNA. Although PCR can be used to amplify small numbers of DNA molecules in low microlitre or nanolitre volumes in vitro, problems are associated with the dilution of heterozygous DNA. This could result in incorrect genotyping results (homozygous genotypes) because of the preferential amplification of one of the alleles. Extreme dilution indicates that there is a sufficiently high integrity of the nucleic-acid templates to allow efficient amplification. Furthermore, amplification of low concentrations of nucleic acids requires strict quality control, as it is often associated with contamination problems, which can be problematic in high-throughput applications. In contrast to nucleic-acid manipulation, it is currently impossible to amplify proteins from a biological sample. R E V I E W S Miniaturization in functional genomicsGenome research and functional genomics have catalysed the development of high-throughput and miniaturized approaches for the analysis of biomolecules [5][6][7][8] . Figure 1 | DNA micorarrays. DNA microarrays that are based on non-porous solid supports such as glass have paved the way for highly parallel miniaturized analysis of biomolecules. Using robotic workstations, ~0.25-1 nl of DNA solution are printed on a slide, creating spots that range from 100 to 150 µm in diameter. Successive samples are then spaced to avoid contact between adjacent spots, with approximately 200 µm between each spot 7 . Currently, about 50,000 cDNAs can be robotically spotted onto a microscope slide and hybridized with a fluorescently labelled probe. Using photolithographical masking techniques, arrays that have 400,000 distinct oligonucleotides have been produced, each in its own 20-µm 2 region (today, this can be as small as ~5 µm 2 ) REF. 8. After spotting, nucleic-acid samples can be hybridized on these pre-structured microarrays. Hybridization events are monitored using fluorescence scanners. A typical fluorescence read-out of highly parallel hybridization on a cDNA microarray is shown in the bottom panel. Box 1 | Technological problems in miniaturizationEvery molecular biological method can be miniaturized from microl...

show abstract

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures

Cited by 79 publications

References 25 publications

Elastase Digests

Elastase Digests

Identification of a Chlorobenzene Reductive Dehalogenase in Dehalococcoides sp. Strain CBDB1

Miniaturization in functional genomics and proteomics

Contact Info

Product

Resources

About