An improved method for tracking and reducing the void volume in nano HPLC?MS with micro trapping columns

Mitulović, Goran; Smoluch, Marek; Chervet, Jean-Pierre; Steinmacher, Ines; Kungl, Andreas J.; Mechtler, Karl

doi:10.1007/s00216-003-2047-2

Cited by 60 publications

(38 citation statements)

References 26 publications

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“…The HPLC systems were configured in a preconcentration setup with a 150-m-inner diameter reverse phase trapping column (53). Peptides were separated on a 75-m-inner diameter reverse phase main column by applying a 40-min binary gradient (solvent A: 0.1% FA; solvent B: 0.1% FA, 84% ACN) ranging from 5 to 95% solvent B at a flow rate of 270 nl/min.…”

Section: Methodsmentioning

confidence: 99%

Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase

Reinders

Wagner

Zahedi

et al. 2007

Molecular & Cellular Proteomics

153

133

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Mitochondria are crucial for numerous cellular processes, yet the regulation of mitochondrial functions is only understood in part. Recent studies indicated that the number of mitochondrial phosphoproteins is higher than expected; however, the effect of reversible phosphorylation on mitochondrial structure and function has only been defined in a few cases. It is thus crucial to determine authentic protein phosphorylation sites from highly purified mitochondria in a genetically tractable organism. The yeast Saccharomyces cerevisiae is a major model organism for the analysis of mitochondrial functions. We isolated highly pure yeast mitochondria and performed a systematic analysis of phosphorylation sites by a combination of different enrichment strategies and mass spectrometry. We identified 80 phosphorylation sites in 48 different proteins. These mitochondrial phosphoproteins are involved in critical mitochondrial functions, including energy metabolism, protein biogenesis, fatty acid metabolism, metabolite transport, and redox regulation. By combining yeast genetics and in vitro biochemical analysis, we found that phosphorylation of a serine residue in subunit g (Atp20) regulates dimerization of the mitochondrial ATP synthase. The authentic phosphoproteome of yeast mitochondria will represent a rich source to uncover novel roles of reversible protein phosphorylation. Molecular & Cellular Proteomics 6:1896 -1906, 2007.Mitochondria are the central organelle for the energy metabolism of eukaryotic cells and are critical for numerous metabolic pathways, including that for amino acids, lipids, heme, and iron-sulfur clusters, and play key roles in the regulation of programmed cell death (1-5). It is evident that these processes have to be tightly regulated to permit a mitochondrial response to changes in energy demand, cellular metabolism, or environmental conditions (6, 7). Until recently the most common regulatory mechanism of eukaryotic cells, reversible phosphorylation (8 -10), was considered to represent an exception in the case of mitochondria, including the E1 subunit of pyruvate dehydrogenase and the branched-chain ␣-ketoacid dehydrogenase (11)(12)(13)(14).A number of recent studies have provided evidence that phosphorylation of mitochondrial proteins is much more frequent than expected (15-23). (i) Incubation of isolated mitochondria with radiolabeled ATP or staining of mitochondrial proteins with phosphospecific dyes suggested that a substantial fraction of mitochondrial proteins are phosphorylated (24 -27). A limitation of these approaches is the inability to identify the specific phosphorylated amino acid residues in addition to the possibility of nonspecific labeling of proteins.(ii) Proteomics analysis of isolated mitochondria by mass spectrometry revealed the presence of numerous protein kinases and phosphatases (28 -34), implying that reversible protein phosphorylation may be a widespread mechanism of regulating mitochondrial function. The most comprehensive proteomics analysis of mitochondria, the PROM...

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Section: Methodsmentioning

confidence: 99%

Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase

Reinders

Wagner

Zahedi

et al. 2007

Molecular & Cellular Proteomics

153

133

View full text Add to dashboard Cite

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“…This will remove the phosphate buffer originating from elution and other impurities. The sample itself will bind to the RP trap column 15 . The sample is injected by using the ''user-defined injection protocol-UDP'': (i) Aspirate 16 26| Elute the sample from the trap column onto the separation nano column and detect with a mass spectrometer.…”

Section: Hplc-ms/ms Analysis Of the Prepared Sample 25|mentioning

confidence: 99%

“…The gradient described starts at 45 min (the trap column is switched online with the separation column) and continues for 30 min. After applying a high organic wash step (95% mobile phase B), the trap column is switched back to offline mode and equilibrated with the loading mobile phase 15 . The MS data are recorded only for the time when both columns are online.…”

Section: Introductionmentioning

confidence: 99%

“…By slowly increasing the amount of mobile phase B in the gradient, a better separation of coeluting peptides is possible. Finally, this approach results in a more sensitive detection in MS. After applying a high organic wash step (95% mobile phase B), the trap column is switched back to offline mode and equilibrated with the loading mobile phase 15 . The MS data are recorded only for the time when both columns are online.…”

Section: Introductionmentioning

confidence: 99%

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Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis

et al. 2006

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We have developed a new offline chromatographic approach for the selective enrichment of phosphorylated peptides that is directly compatible with subsequent analysis by online nano electrospray ionization tandem mass spectrometry. In this technique, a titanium dioxide (TiO 2 )-packed pipette tip is used as a phosphopeptide trap that acts as an offline first-dimension separation step in a twodimensional chromatography system. This is followed by online nano reversed-phase high-performance liquid chromatography. Here, we present suitable methods for enrichment, optimized separately for each step: sample loading, washing and elution from the TiO 2 -filled tips. To increase the trapping selectivity of the TiO 2 column, we used the sodium salt of 1-octanesulfonic acid combined with 2,5-dihydroxybenzoic acid as ion-pairing agents and displacers for acidic peptides. These agents also improve the binding of phosphorylated peptides and block the binding of non-phosphorylated ones. This enrichment procedure takes 30 min, followed by a 100-min HPLC program, including washing and an elution gradient. INTRODUCTIONThe majority of biochemical processes are induced and controlled by post-translational modifications on certain proteins. One of the most frequent modifications is phosphorylation of the amino acids-serine, threonine and tyrosine. It is this reversible phosphorylation that regulates the major (or even the majority of) cellular processes, and it has been estimated that almost 30% of all proteins in mammalian cells are phosphorylated at some point during their expression 1,2 . This alone would be reason enough to invest time and resources in the analysis of phosphopeptides.Despite the fact that there are so many phosphoproteins in the cell, phosphorylated residues remain at very low concentrations physiologically and are present in substoichiometric quantities. The presence of high-abundance peptides in samples of biological origin makes it necessary to develop an efficient separation and enrichment method for phosphopeptides.Earlier established methods for phosphorylation site detection relied on Edman degradation of 32 P-labeled peptides. Edman degradation is a robust and well-established method, but it shows some limitations when analyzing complex samples and cannot fulfill the requirements for sensitive detection of low concentrations of phosphopeptides [1][2][3] . Besides the additional needed labeling of the phosphate residues, it would take more than a hundred fold in time to chemically treat and analyze a complex sample related to other techniques for sequencing and phosphorylation site mapping.Identifying phosphorylation sites in different proteins through mass spectrometry (MS) requires a proper pre-separation and enrichment of the phosphopeptides performed online by the HPLC, because of the often highly complex sample composition.Some phosphopeptide-selective techniques have been developed, such as immobilized metal affinity chromatography (IMAC) [4][5][6] or two-dimensional HPLC systems using strong cation...

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“…Therefore, LC-MS-systems and especially nano-LC-MS/ MS have been widely used on account for their high sensitivity (lower femtomole-range), good reproducibility and convenient automation [60]. Furthermore, larger sample volumes (up to 100 mL) can be applied using a precolumn concentration method as described by Mitulovic et al [61]. Thereby, the sample is flushed onto a microscale precolumn using high flow rates (5-50 mL/min).…”

Section: Localization Of Phosphorylation Sitesmentioning

confidence: 99%

State-of-the-art in phosphoproteomics

2005

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Presently, phosphorylation of proteins is the most studied and best understood PTM. However, the analysis of phosphoproteins and phosphopeptides is still one of the most challenging tasks in contemporary proteome research. Since not every phosphoprotein is accessible by a certain method and identification of the phosphorylated amino acid residue is required in the majority of cases, various strategies for the detection and localization of phosphorylations have been developed. Identification and localization of protein phosphorylations is mostly done by MS nowadays but phosphoproteins and -peptides are often suppressed in comparison to the unphosphorylated species if measured in complex mixtures. Thus, the isolation of pure phosphopeptide samples is a main task. This review gives an overview over the most frequently used methods in isolation and detection of phosphoproteins and -peptides such as specific enrichment or separation strategies as well as the localization of the phosphorylated residues by various mass spectrometric techniques.

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An improved method for tracking and reducing the void volume in nano HPLC?MS with micro trapping columns

Cited by 60 publications

References 26 publications

Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase

Profiling Phosphoproteins of Yeast Mitochondria Reveals a Role of Phosphorylation in Assembly of the ATP Synthase

Titanium dioxide as a chemo-affinity solid phase in offline phosphopeptide chromatography prior to HPLC-MS/MS analysis

State-of-the-art in phosphoproteomics

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