Principles of Electrospray Ionization

Wilm, Matthias

doi:10.1074/mcp.m111.009407

Cited by 229 publications

(117 citation statements)

References 26 publications

(48 reference statements)

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“…Consequently, for optimal results both instruments have to operate under perfect conditions, and the methods used on both instruments have to be adjusted to each other. Joining is achieved by ionization of the peptides with electrospray ionization (ESI) 19 . The flow rate of the electrosprayed solvent is closely related to the ionization efficiency, with flow rates in the order of 10 nl min − 1 yielding the highest mass spectrometric sensitivity 20,21 .…”

Section: Quality Control Samplementioning

confidence: 99%

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Köcher

Pichler

Swart³

et al. 2012

Nat Protoc

109

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The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 10 h of nanoLC-MS/MS measurement time.

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Section: Quality Control Samplementioning

confidence: 99%

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Köcher

Pichler

Swart³

et al. 2012

Nat Protoc

109

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“…Electro spray ionization (ESI) mass spectrometry (MS) analysis of the final product was performed by M-scan (West Chester, PA) [29].…”

Section: Mass Spectrometrymentioning

confidence: 99%

Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG

Buyel

Bautista

Fischer

et al. 2012

Journal of Chromatography B

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“…Electrospray ionization (ESI) ionizes molecules directly from the liquid phase 5 ; matrix-assisted laser desorption ionization (MALDI) requires that the biomolecules are co-crystallized with ultraviolet-absorbing organic molecules (i.e. matrix molecules) 6 .…”

Section: Introductionmentioning

confidence: 99%

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Signor

Erba

2013

JoVE

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Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. C labelling).Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

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Principles of Electrospray Ionization

Cited by 229 publications

References 26 publications

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Extraction, purification and characterization of the plant-produced HPV16 subunit vaccine candidate E7 GGG

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

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