Ultraviolet laser desorption/ionization mass spectrometry of proteins above 100,000 daltons by pulsed ion extraction time-of-flight analysis

Spengler, Bernhard; Cotter, Robert J.

doi:10.1021/ac00207a004

Cited by 154 publications

(57 citation statements)

References 21 publications

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“…, such method yields high sensitivity when a biomolecule mass is above 100 kDa. Remarkable analyses of intact proteins were carried out using home-made instruments between the end of the 1980's and the beginning of the 1990's [8][9][10][11] . MALDI-TOF can be also used as a screening tool to evaluate the quality of protein samples because it requires less time for sample preparation and is less susceptible to interferences due to common impurities (e.g.…”

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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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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“…With respect to mass spectrometry these studies focused on either direct laser desorption/ionization (LDI) with pulsed infrared (IR) and ultraviolet (UV) lasers, e.g., [7][8][9][10][11][12][13][14][15] or, more relevant for this work, MALDI-MS [5, 16 -28] with pulsed ultraviolet lasers. The first measurements of initial ion velocities in infrared MALDI were reported only very recently by Ermer et al [29].…”

mentioning

confidence: 99%

“…In the field-free drift methods (FFDM) initial velocities have been deduced either purely from the drift time in a field-free region [29], a comparison between the flight times with and without a field in a first ion stage region [18,19], or in orthogonal acceleration geometries in which ions were extracted perpendicular to their centre of mass movement, after passing through a field-free region [15,20]. In yet another variant initial ion velocities were determined in a modified Wiley-McLaren set-up [23].…”

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confidence: 99%

Measurements of mean initial velocities of analyte and matrix ions in infrared matrix-assisted laser desorption ionization mass spectrometry

Berkenkamp

Menzel

Hillenkamp

et al. 2002

J. Am. Soc. Mass Spectrom.

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The mean initial velocities of analyte ions ranging in molecular weight from 1000 Da to 150 kDa and desorbed with a pulsed Er:YAG laser from various solid-state and liquid IR MALDI matrices were measured along with those of the matrix ions. Experiments with UV MALDI were performed for comparison in addition for a 2,5-dihydroxybenzoic acid preparation. Two different measurement principles were employed, (1) a delayed extraction method, relying on the initial velocity-dependent increase of flight times with delay time between laser and HV ion extraction pulse, and (2) a field-free drift method in which the first region of a two-stage ion source was varied in length and the flight times compared. The two methods yielded somewhat different values for the mean initial ion velocities. Based on a detailed discussion of the measurement principles it is suggested that the actual initial velocities of IR MALDI ions lie between the limits set by the two methods. The influences of the analyte-to-matrix ratio, laser fluence, and laser wavelength on the initial ion velocities were also investigated. Significant differences between the desorption mechanisms for liquid and solid-state matrices were observed. M easurements of the initial kinetic energy of ions and particles form a straightforward approach for investigations on the mechanistics of laser desorption and laser desorption/ionization processes. In time-of-flight (TOF) mass spectrometry, the large and mass-dependent initial kinetic energies of analyte ions in matrix-assisted laser desorption ionization (MALDI), along with a large width in the energy distributions have moreover posed substantial limitations on the instrumental performance for a long time. With state-of-the-art delayed extraction (DE) [1,2] instruments these problems can now partly be overcome [3,4], although sophisticated calibration procedures incorporating explicit initial velocity values are required [5,6]. The determination of the initial velocities of MALDI ions has, therefore, found substantial interest in the last years also from this practical viewpoint.A relatively large number of studies on the kinetic energies of laser desorbed* particles or ions have been performed in the past. With respect to mass spectrometry these studies focused on either direct laser desorption/ionization (LDI) with pulsed infrared (IR) and ultraviolet (UV) lasers, e.g., [7][8][9][10][11][12][13][14][15] or, more relevant for this work, MALDI-MS [5, 16 -28] with pulsed ultraviolet lasers. The first measurements of initial ion velocities in infrared MALDI were reported only very recently by Ermer et al. [29].Different experimental approaches for the determination of initial (ion) velocities have been pursued in these studies. A survey of the employed techniques for UV MALDI has recently been given by Glü ckmann and Karas [17]. Following their classification, the techniques can essentially be arranged into three groups.In the so-called delayed extraction methods (DEM) [5,16,17], the mean initial axial velocity is deduced...

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“…Protein identification by linear MALDI-TOF [1][2][3] relies mostly on PMF [4][5][6][7][8]. However, these instruments, when equipped with ion mirrors (i.e.…”

Section: Introductionmentioning

confidence: 99%

Parallel post‐source decay for increasing protein identification confidence levels from 2‐D gels

et al. 2008

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Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.

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Ultraviolet laser desorption/ionization mass spectrometry of proteins above 100,000 daltons by pulsed ion extraction time-of-flight analysis

Cited by 154 publications

References 21 publications

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

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

Measurements of mean initial velocities of analyte and matrix ions in infrared matrix-assisted laser desorption ionization mass spectrometry

Parallel post‐source decay for increasing protein identification confidence levels from 2‐D gels

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