Improved in‐gel approaches to generate peptide maps of integral membrane proteins with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Montfort, Bart A. van; Cañas, Benito; Duurkens, Ria H.; Godovac‐Zimmermann, Jasminka; Robillard, George T.

doi:10.1002/jms.288

Cited by 75 publications

(72 citation statements)

References 28 publications

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“…Sample aliquots were cocrystallized with the matrix on the MALDI-TOF sample plate at various dilutions, and the dried residues were washed twice with cold 0.1% aqueous trifluoroacetic acid to remove salts and residual glutaraldehyde. A volume of 0.5 l of 0.1% aqueous octylglucoside was added to each residue, followed by evaporation under ambient laboratory conditions (46), as this was found to improve the mass spectra.…”

Section: Methodsmentioning

confidence: 99%

CsrA Regulates Translation of the Escherichia coli Carbon Starvation Gene, cstA , by Blocking Ribosome Access to the cstA Transcript

Dubey¹,

Baker²,

et al. 2003

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CsrA is a global regulator that binds to two sites in the glgCAP leader transcript, thereby blocking ribosome access to the glgC Shine-Dalgarno sequence. The upstream CsrA binding site (GCACACGGAU) was used to search the Escherichia coli genomic sequence for other genes that might be regulated by CsrA. cstA contained an exact match that overlapped its Shine-Dalgarno sequence. cstA was previously shown to be induced by carbon starvation and to encode a peptide transporter. Expression of a cstA-lacZ translational fusion in wild-type and csrA mutant strains was examined. Expression levels in the csrA mutant were approximately twofold higher when cells were grown in Luria broth (LB) and 5-to 10-fold higher when LB was supplemented with glucose. It was previously shown that cstA is regulated by the cyclic AMP (cAMP)-cAMP receptor protein complex and transcribed by ⌭ 70 . We investigated the influence of S on cstA expression and found that a S deficiency resulted in a threefold increase in cstA expression in wild-type and csrA mutant strains; however, CsrA-dependent regulation was retained. The mechanism of CsrA-mediated cstA regulation was also examined in vitro. Cross-linking studies demonstrated that CsrA is a homodimer. Gel mobility shift results showed that CsrA binds specifically to cstA RNA, while coupled-transcription-translation and toeprint studies demonstrated that CsrA regulates CstA synthesis by inhibiting ribosome binding to cstA transcripts. RNA footprint and boundary analyses revealed three or four CsrA binding sites, one of which overlaps the cstA ShineDalgarno sequence, as predicted. These results establish that CsrA regulates translation of cstA by sterically interfering with ribosome binding.

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Section: Methodsmentioning

confidence: 99%

CsrA Regulates Translation of the Escherichia coli Carbon Starvation Gene, cstA , by Blocking Ribosome Access to the cstA Transcript

Dubey¹,

Baker²,

et al. 2003

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“…The spots then were washed and digested with modified trypsin (Promega, Madison, WI) (Shevchenko et al, 1996) or pepsin, and peptides extracted automatically using the ProGest robot (Genomic Solutions). The acetone-soluble proteins were digested with trypsin and chymotrypsin or cleaved by limited acid hydrolysis (Shevchenko et al, 2000) or CnBr (van Montfort et al, 2002). Fractions from off-line RP-HPLC were digested in-solution with pepsin (in 70% formic acid and 1 mM HCl), trypsin (in 8 M urea), or chymotrypsin (in 8 M urea).…”

Section: Ms Analysismentioning

confidence: 99%

In-Depth Analysis of the Thylakoid Membrane Proteome ofArabidopsis thalianaChloroplasts: New Proteins, New Functions, and a Plastid Proteome Database[W]

et al. 2004

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An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one-and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were a-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http:// cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

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“…Two other labeled proteins of ca. 20 and 55 kDa were observed but could not be identified by MS. Possible reasons for the inability to identify these proteins include resistance to trypsin digestion (7,40), the mass-altering effect of biotinylation, or the presence of unknown posttranslational modifications, resulting in a failure to derive peptide mass matches. However, the 20-kDa protein demonstrated a significant degree of surface labeling and may therefore represent an additional as-yet-uncharacterized, surface-exposed protein.…”

Section: Vol 73 2005mentioning

confidence: 99%

Surfaceome of Leptospira spp

et al. 2005

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The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31 LipL45 , GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<10 7 cells) offering the potential to analyze bacterial surface expression during infection.

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Improved in‐gel approaches to generate peptide maps of integral membrane proteins with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Cited by 75 publications

References 28 publications

CsrA Regulates Translation of the Escherichia coli Carbon Starvation Gene, cstA , by Blocking Ribosome Access to the cstA Transcript

CsrA Regulates Translation of the Escherichia coli Carbon Starvation Gene, cstA , by Blocking Ribosome Access to the cstA Transcript

In-Depth Analysis of the Thylakoid Membrane Proteome ofArabidopsis thalianaChloroplasts: New Proteins, New Functions, and a Plastid Proteome Database[W]

Surfaceome of Leptospira spp

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