MALDI MS profiling of post-DRE urine samples highlights the potential of b-Microseminoprotein as a marker for prostatic diseases

BACKGROUND. To use spectra acquired by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) from pre- and post-digital rectal examination (DRE) urine samples to search for discriminating peaks that can adequately distinguish between benign and malignant prostate conditions, and identify the peaks’ underlying biomolecules. METHODS. Twenty-five participants with prostate cancer (PCa) and 27 participants with a variety of benign prostatic conditions as confirmed by a 10-core tissue biopsy were included. Pre- and post-DRE urine samples were prepared for MALDI MS profiling using an automated clean-up procedure. Following mass spectra collection and processing, peak mass and intensity were extracted and subjected to statistical analysis to identify peaks capable of distinguishing between benign and cancer. Logistic regression was used to combine markers to create a sensitive and specific test. RESULTS. A peak at m/z 10,760 was identified as b-microseminoprotein (b-MSMB) and found to be statistically lower in urine from PCa participants using the peak’s average areas. By combining serum prostate-specific antigen (PSA) levels with MALDI MS-measured b-MSMB levels, optimum threshold values obtained from Receiver Operator characteristics curves gave an increased sensitivity of 96% at a specificity of 26%. CONCLUSIONS. These results demonstrate that with a simple sample clean-up followed by MALDI MS profiling, significant differences of MSMB abundance were found in post-DRE urine samples. In combination with PSA serum levels, obtained from a classic clinical assay led to high classification accuracy for PCa in the studied sample set. Our results need to be validated in a larger multicenter prospective randomized clinical trial.

Enhanced MALDI MS sensitivity by weak base additives and glycerol sample coating

The concept of rationally designing MALDI matrices has been extended to the next “whole sample” level. These studies have revealed some unexpected and exploitable insights in improving MALDI sensitivity. It is shown that (i) additives which only provide additional laser energy absorption are best to be avoided; (ii) the addition of proton donors in the form of protonated weak bases can be highly beneficial; (iii) the addition of glycerol for coating crystalline samples is highly recommended. Overall, analytical sensitivity has been significantly increased compared to the current “gold” standards in MALDI MS, and new insights into the mechanisms and processes of MALDI have been gained.

Ionic liquids and other liquid matrices for sensitive MALDI MS analysis

Although liquid matrix-assisted laser desorption/ionization (MALDI) has been used in mass spectrometry (MS) since the early introduction of MALDI, its substantial lack of sensitivity compared to solid (crystalline) MALDI was for a long time a major hurdle to its analytical competitiveness. In the last decade, this situation has changed with the development of new sensitive liquid matrices, which are often based on a binary matrix acid/base system. Some of these matrices were inspired by the recent progress in ionic liquid research, while others were developed from revisiting previous liquid MALDI work as well as from a combination of these two approaches. As a result, two high-performing liquid matrix classes have been developed, the ionic liquid matrices (ILMs) and the liquid support matrices (LSMs), now allowing MS measurements at a sensitivity level that is very close to the level of solid MALDI and in some cases even surpasses it. This chapter provides some basic information on a selection of highly successful representatives of these new liquid matrices and describes in detail how they are made and applied in MALDI MS analysis.

Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS

Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-tovacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental setup and optimization strategy is described for liquid AP-MALDI MS which improves the ionization effi- ciency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2 fmol/lL (0.5 lL, i.e. 1 fmol, deposited on the target) with very low sample consumption in the low nL-range.

Liquid matrix deposition on conductive hydrophobic surfaces for tuning and quantitation in UV-MALDI mass spectrometry

With its highly fluctuating ion production matrix-assisted laser desorption/ionization (MALDI) poses many practical challenges for its application in mass spectrometry. Instrument tuning and quantitative ion abundance measurements using ion signal alone depend on a stable ion beam. Liquid MALDI matrices have been shown to be a promising alternative to the commonly used solid matrices. Their application in areas where a stable ion current is essential has been discussed but only limited data have been provided to demonstrate their practical use and advantages in the formation of stable MALDI ion beams. In this article we present experimental data showing high MALDI ion beam stability over more than two orders of magnitude at high analytical sensitivity (low femtomole amount prepared) for quantitative peptide abundance measurements and instrument tuning in a MALDI Q-TOF mass spectrometer. Samples were deposited on an inexpensive conductive hydrophobic surface and shrunk to droplets <10 nL in size. By using a sample droplet <10 nL it was possible to acquire data from a single irradiated spot for roughly 10,000 shots with little variation in ion signal intensity at a laser repetition rate of 5-20 Hz.

Investigation of liquid MALDI and optimization for instrument tuning and quantitative measurements

The success of Matrix-assisted laser desorption / ionisation (MALDI) in fields such as proteomics has partially but not exclusively been due to the development of improved data acquisition and sample preparation techniques. This has been required to overcome some of the short comings of the commonly used solid-state MALDI matrices such as - cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). Solid state matrices form crystalline samples with highly inhomogeneous topography and morphology which results in large fluctuations in analyte signal intensity from spot to spot and positions within the spot. This means that efficient tuning of the mass spectrometer can be impeded and the use of MALDI MS for quantitative measurements is severely impeded. Recently new MALDI liquid matrices have been introduced which promise to be an effective alternative to crystalline matrices. Generally the liquid matrices comprise either ionic liquid matrices (ILMs) or a usually viscous liquid matrix which is doped with a UV lightabsorbing chromophore [1-3]. The advantages are that the droplet surface is smooth and relatively uniform with the analyte homogeneously distributed within. They have the ability to replenish a sampling position between shots negating the need to search for sample hot-spots. Also the liquid nature of the matrix allows for the use of additional additives to change the environment to which the analyte is added.

Liquid AP-UV-MALDI enables stable ion yields of multiply charged peptide and protein ions for sensitive analysis by mass spectrometry

In biological mass spectrometry (MS), two ionization techniques are predominantly employed for the analysis of larger biomolecules, such as polypeptides. These are nano-electrospray ionization [1, 2] (nanoESI) and matrix-assisted laser desorption/ionization [3, 4] (MALDI). Both techniques are considered to be “soft”, allowing the desorption and ionization of intact molecular analyte species and thus their successful mass-spectrometric analysis. One of the main differences between these two ionization techniques lies in their ability to produce multiply charged ions. MALDI typically generates singly charged peptide ions whereas nanoESI easily provides multiply charged ions, even for peptides as low as 1000 Da in mass. The production of highly charged ions is desirable as this allows the use of mass analyzers, such as ion traps (including orbitraps) and hybrid quadrupole instruments, which typically offer only a limited m/z range (< 2000–4000). It also enables more informative fragmentation spectra using techniques such as collisioninduced dissociation (CID) and electron capture/transfer dissociation (ECD/ETD) in combination with tandem MS (MS/MS). [5, 6] Thus, there is a clear advantage of using ESI in research areas where peptide sequencing, or in general, the structural elucidation of biomolecules by MS/MS is required. Nonetheless, MALDI with its higher tolerance to contaminants and additives, ease-of-operation, potential for highspeed and automated sample preparation and analysis as well as its MS imaging capabilities makes it an ionization technique that can cover bioanalytical areas for which ESI is less suitable. [7, 8] If these strengths could be combined with the analytical power of multiply charged ions, new instrumental configurations and large-scale proteomic analyses based on MALDI MS(/MS) would become feasible.

AP-UV-MALDI mit flüssigen Matrizes: stabile Ionenausbeuten von mehrfach geladenen Peptid- und Proteinionen für die empfindliche Massenspektrometrie

In der biologischen Massenspektrometrie (MS) werden überwiegend zwei Ionisationstechniken für die Analyse von grçßeren Biomolekfürlen wie Polypeptiden eingesetzt. Dies sind die Nano-Elektrospray-Ionisation[1,2] (nanoESI) und die matrixunterstfürtzte Laserdesorption/-ionisation[3, 4] (MALDI). Beide Techniken werden als „sanft“ bezeichnet, weil sie die Desorption und Ionisation von intakten Analytmolekfürlen und damit ihre erfolgreiche massenspektrometrische Analyse erlauben. Einer der wichtigsten Unterschiede zwischen diesen beiden Ionisationstechniken liegt in ihrer F�higkeit, mehrfach geladene Ionen zu erzeugen. MALDI erzeugt typischerweise einfach geladene Peptidionen, w�hrend nano- ESI leicht mehrfach geladene Ionen produziert, sogar für Peptide mit einer Masse von weniger als 1000 Da. Die Erzeugung von hoch geladenen Ionen ist wünschenswert, da dies die Verwendung von Massenanalysatoren wie Ionenfallen (inkl. Orbitraps) und Hybrid-Quadrupolinstrumenten ermçglicht, die typischerweise nur einen begrenzten m/z- Bereich (<2000–4000) bieten. Hohe Ladungszust�nde ermçglichen auch die Aufnahme von informativeren Fragmentionenspektren, wenn Methoden wie die kollisionsinduzierte Dissoziation (CID), die Elektroneneinfang-Dissoziation (ECD) und die Elektronentransfer-Dissoziation (ETD) in Kombination mit der Tandem-MS (MS/MS) verwendet werden.