Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Miliotis, Tasso; Ericsson, Per; Markó-Varga, György; Svensson, Robert; Nilsson, Johan; Laurell, Thomas; Bischoff, Rainer

doi:10.1016/s0378-4347(00)00458-8

Cited by 36 publications

(29 citation statements)

References 23 publications

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“…Micro-liquid chromatography: Micro-liquid chromatography (micro-LC) using capillary-column formats is an attractive technique for separation problems that require low sample loads and mass-spectrometric interfacing. [5] These needs apply in the proteomics area in which sample volumes are strongly limited but the demand for detection sensitivity and selectivity is high. To shrink the imprinted polymers to match the micro-LC format, we used our recently reported technique based on grafting imprinted polymer layers onto flow-through polyA C H T U N G T R E N N U N G (TRIM) monoliths with unreacted surface double bonds as anchor points.…”

Section: Wwwchemeurjorgmentioning

confidence: 99%

“…Comprehensive mapping of the phosphoproteome is, therefore, an important objective in todays proteomics research. [2][3][4][5] In this context, it has proven particularly difficult to obtain a comprehensive view of proteins phosphorylated at tyrosine (Scheme 1). [4] This is because the prefractionation techniques used for this purpose commonly lack the selectivity, sensitivity, and robustness required for reproducibly extracting these low-abundant proteins.…”

Section: Introductionmentioning

confidence: 99%

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A Phosphotyrosine‐Imprinted Polymer Receptor for the Recognition of Tyrosine Phosphorylated Peptides

Emgenbroich¹,

Borrelli²,

Shinde³

et al. 2008

Chemistry A European J

114

125

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Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.

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Section: Wwwchemeurjorgmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Phosphotyrosine‐Imprinted Polymer Receptor for the Recognition of Tyrosine Phosphorylated Peptides

Emgenbroich¹,

Borrelli²,

Shinde³

et al. 2008

Chemistry A European J

114

125

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“…Consequently, by moving the SPE-bed into the microdispenser as close to the orifice as possible, this may be accomplished as is shown in Fig. 6B, a natural course, in line with our earlier development of chip-based SPE [ 48,49] and on-spot sample enrichment [41,54]. Design and principle of operation of the SPE-microdispenser is described in Fig.…”

Section: Sample Preparation -Microstructure Utilizationmentioning

confidence: 68%

“…Recent developments within our research group have shown to generate highly similar MS spectra even after several months of MALDI-crystal storage [40]. We could conclude that this is applicable not only to proteins in general but especially to phosphopeptides where the phospho-stoichiometry determination is of key interest [41].…”

Section: Liquid-phase Multidimensional Chromatography Technologiesmentioning

confidence: 77%

New directions of miniaturization within the proteomics research area

2003

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An overview of the current trends within protein expression profiling is given where multidimensional separation of both gel‐ and liquid‐phase techniques linked to mass spectrometry is viewed as a major route in the global proteome mapping. A clear trend in these biochemical developments is the effort to sequence and identify low‐abundant protein expressions where assay miniaturization and integrated sample processing plays a central role. Two areas of miniaturization within the proteomics field are addressed: (i) sample cleanup and enrichment, and (ii) silicon microstructure developments for protein chip microarrays.

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“…For optimum separation, 0.1% TFA is commonly added to the mobile phase in RP LC of peptides and proteins. Since it is well known that the combination of TFA and ESI MS results in signal reduction and spray instability [18][19][20], a combination of formic acid (0.1%) and TFA (0.01%) was used as additive in the mobile phase. Several different solvent gradients were evaluated in order to optimize the separation of the tryptic peptides.…”

Section: Column Switching Capillary Lc Esi Tof Msmentioning

confidence: 99%

Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

Pepaj¹,

Holm

Fleckenstein

et al. 2006

J of Separation Science

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Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometryIn the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.

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Analysis of regulatory phosphorylation sites in ZAP-70 by capillary high-performance liquid chromatography coupled to electrospray ionization or matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Cited by 36 publications

References 23 publications

A Phosphotyrosine‐Imprinted Polymer Receptor for the Recognition of Tyrosine Phosphorylated Peptides

A Phosphotyrosine‐Imprinted Polymer Receptor for the Recognition of Tyrosine Phosphorylated Peptides

New directions of miniaturization within the proteomics research area

Fractionation and separation of human salivary proteins by pH‐gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

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