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Employing ‘Second Generation’ Matrices

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Book cover Advances in MALDI and Laser-Induced Soft Ionization Mass Spectrometry

Abstract

The ‘first generation’ of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) matrices was found randomly during the early days of MALDI by empirical testing of hundreds of small molecules with molecular weights of typically about 150–250 g/mol and high absorption at the wavelength of the laser used for irradiation. For the ‘second generation’ matrices the structures of established matrix molecules were systematically modified by varying the nature, number, and position of their functional groups. The objective was to gain a better understanding of how the physicochemical processes essential for matrix and analyte ion generation are affected by the matrix molecular structure. With the uncovering of key ionization steps, predictions regarding the MALDI performance of in-silico designed matrix compounds came within reach by computational calculations. This marked a milestone in matrix development and provided valuable information for the creation of optimized compounds. The most comprehensive modifications were done on the core structure of the most widely used matrix α-cyano-4-hydroxycinnamic acid (CHCA) as chemical lead. Three derivatives proved to be outstanding and found their way in different fields of application. The Cl-substituted derivative of CHCA, 4-chloro-α-cyanocinnamic acid (ClCCA), was selected as the most potent matrix for the analysis of several substance classes including peptides. Compared to the hitherto favored CHCA, this new matrix is superior in detecting small amounts of in-solution as well as in-gel digested proteins leading to typically higher sequence coverages. Due to its higher protonation efficiency, discrimination of less basic peptides is strongly diminished which enables more uniform peptide detection. In addition to the more sensitive analysis of acidic peptides, the higher sensitivity also allows for the detection of low-abundant peptides such as phosphopeptides, enzymatically digested peptides with higher numbers of missed cleavages or less or even nonspecific cleavage sites (e.g., generated by elastase, slymotrypsin or proteinase K). This matrix can also be used for the analysis of substance classes such as lipids in positive ion mode and labile glycans in negative ion mode. Another CHCA derivative, α-cyano-2,4-difluorocinnamic acid (DiFCCA), was successfully applied for the most sensitive production of positive ions from phosphatidylcholines. In negative ion mode, a third derivative, α-cyano-4-phenylcinnamic acid amide (Ph-CCA-NH2), showed promising results in lipid analysis. Finally, 1,8-bis(dimethylamino)naphthalene (DMAN) as a very strong base is predestined for negative ionization of acidic compounds. This matrix only generates its intact protonated form [matrix + H]+ and is suitable for the analysis of small molecules in positive and negative ion mode.

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Notes

  1. 1.

    In the case of liquid MALDI samples analytes are naturally well-incorporated into an excess of matrix (cf. the chapters on liquid MALDI MS).

  2. 2.

    Note that hydrogens of phenolic hydroxyl groups can be abstracted even more easily as will be discussed later with the example of CHCA .

  3. 3.

    Note that as mentioned in Sect. 1 the sample morphology can have a large effect on the overall MALDI performance. Wiangnon and Cramer have reported that improvements in peptide ion signal intensity and suppression as published earlier for ClCCA are dependent on the use of the MALDI target plate and were not obtained with AnchorChip target plates (Bruker), which lead to markedly different sample morphologies compared to preparations on normal steel target plates (Wiangnon and Cramer 2015).

  4. 4.

    Note that the combination of both substances leads to homogenous crystallization, a prerequisite for MALDI imaging.

  5. 5.

    ‘Proton sponges’ can contaminate MS instruments with the effect of reduced signal intensities in subsequent positive ion mode measurements. Therefore, ‘proton sponges’ need to be carefully employed in mass spectrometry.

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Authors and Affiliations

  1. Institute for Pharmaceutical Chemistry, University of Frankfurt, Max-von-Laue Str. 9, Frankfurt, 60438, Germany

    U. Bahr

  2. Dr. Franz Köhler Chemie, Werner-von-Siemens-Str. 22-28, Bensheim, 64625, Germany

    T. W. Jaskolla

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  1. U. Bahr
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  2. T. W. Jaskolla
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Correspondence to U. Bahr .

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Editors and Affiliations

  1. Department of Chemistry, University of Reading, Reading, Berkshire, United Kingdom

    Rainer Cramer

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Dedication In Memory of Franz Hillenkamp

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Bahr, U., Jaskolla, T.W. (2016). Employing ‘Second Generation’ Matrices. In: Cramer, R. (eds) Advances in MALDI and Laser-Induced Soft Ionization Mass Spectrometry. Springer, Cham. https://doi.org/10.1007/978-3-319-04819-2_1

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