Fragmentation of leucine enkephalin as a function of laser fluence in a MALDI TOF-TOF.

The effects of laser fluence on ion formation in MALDI were studied using a tandem TOF mass spectrometer with a Nd-YAG laser and alpha-cyano hydrocinnamic acid matrix. Leucine enkephalin ionization and fragmentation were followed as a function of laser fluence ranging from the threshold of ion formation to the maximum available, that is, about 280-930 mJ/mm2. The most notable finding was the appearance of immonium ions at fluence values close to threshold, increasing rapidly and then tapering in intensity with the appearance of typical backbone fragment ions. The data suggest the presence of two distinct environments for ion formation. One is associated with molecular desorption at low values of laser fluence that leads to extensive immonium ion formation. The second becomes dominant at higher fluences, is associated initially with backbone type fragments, but, at the highest values of fluence, progresses to immonium fragments. This second environment is suggestive of ion desorption from large pieces of material ablated from the surface. Arrhenius rate law considerations were used to estimate temperatures associated with the onset of these two processes.

Analysis and Quantitation of Glycated Hemoglobin by Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry.

Measurement of glycated hemoglobin is widely used for the diagnosis and monitoring of diabetes mellitus. Matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometry (MS) analysis of patient samples is used to demonstrate a method for quantitation of total glycation on the ß-subunit of hemoglobin. The approach is accurate and calibrated with commercially available reference materials. Measurements were linear (R(2) > 0.99) across the clinically relevant range of 4% to 20% glycation with coefficients of variation of ≤ 2.5%. Additional and independent measurements of glycation of the α-subunit of hemoglobin are used to validate ß-subunit glycation measurements and distinguish hemoglobin variants. Results obtained by MALDI-TOF MS were compared with those obtained in a clinical laboratory using validated HPLC methodology. MALDI-TOF MS sample preparation was minimal and analysis times were rapid making the method an attractive alternative to methodologies currently in practice.

Tandem time-of-flight mass spectrometry.

A new tandem time-of-flight (TOF-TOF) instrument has been developed by modifying a standard matrix-assisted laser desorption ionization (MALDI)-TOF instrument to make high-performance, high-energy collision-induced dissociation (CID) MALDI tandem mass spectrometry (MS) a practical reality. To optimize fragment spectra quality, the selected precursor ion is decelerated before entering a floating collision cell and the potential difference between the source and the collision cell defines the collision energy of the ions. Standard operating conditions for tandem MS use a 1-kV collision energy with single-collision conditions and increased laser power for ion formation. Hence, both high- and low-energy fragments are observed in MALDI TOF-TOF spectra. On standard peptides, sensitivities down to 1 fmol are demonstrated. On a mixture of two solution tryptic digests at the 25-fmol level, 23 spectra were sufficient to result in proper database identification.

De novo sequencing of peptides using MALDI/TOF-TOF.

The recently developed MALDI TOF-TOF instrument yields relatively complex but interpretable fragmentation spectra. When coupled with a straightforward sequence extension algorithm, it is possible to develop complete peptide sequences de novo from the spectra. This approach has been applied to a set of peptides derived from typtic digestion of electrophoretically separated sea urchin egg membrane proteins. When directed to proteins that have been described previously, the results were in essential agreement with those obtained by conventional data base searching approaches, with certain important exceptions. The present method detected errors in published sequences and was able to develop sequences from peptides differing in mass by one dalton (Da). These results show both the power of the present approach and the need for using de novo methods more frequently than may be otherwise appreciated.

The future of biological mass spectrometry.

Biological applications of mass spectrometry have grown exponentially since the discovery of MALDI and electrospray ionization techniques. This growth has been further fueled by the massive volume of DNA sequence information that is now available. An ambitious goal of some of this research is to monitor the level and modification of all proteins and metabolites in a biological sample such as plasma. A major research effort in mass spectrometry and related disciplines has been expended over the past several years toward reaching this and other less ambitious goals, and considerable progress has been made; but the presently available tools are clearly not sufficient for these very difficult tasks. In this "critical insight" discussion we suggest that recent advances in time-of-flight (TOF) technology with MALDI ionization may provide some important new tools for achieving the goals of biological research.

Modern MALDI time-of-flight mass spectrometry.

This paper focuses on development of time-of-flight (TOF) mass spectrometry in response to the invention of matrix-assisted laser desorption/ionization (MALDI). Before this breakthrough ionization technique for nonvolatile molecules, TOF was generally considered as a useful tool for exotic studies of ion properties but was not widely applied to analytical problems. Improved TOF instruments and software that allow the full potential power of MALDI to be applied to difficult biological applications are described. A theoretical approach to the design and optimization of MALDI-TOF instruments for particular applications is presented. Experimental data are provided that are in excellent agreement with theoretical predictions of resolving power and mass accuracy. Data on sensitivity and dynamic range using kilohertz laser rates are also summarized. These results indicate that combinations of high-performance MALDI-TOF and TOF-TOF with off-line high-capacity separations may ultimately provide throughput and dynamic range several orders of magnitude greater than those currently available with electrospray LC-MS and MS-MS.

Novel three-dimensional MALDI plate for interfacing high-capacity LC separations with MALDI-TOF.

Novel matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) sample plates employing collimated-hole structures have been developed that allow capture and concentration of samples while simultaneously acting as a sink for carrier solvents. These plates were designed to provide an efficient interface between higher-capacity liquid chromatography (LC) separations and MALDI-TOF mass spectrometry (MS). LC-MALDI using conventional plates can accommodate the low-flow (< 1 microL/min) separation protocols typically used in on-line LC-MS methods, and can also be used with higher flow rate, larger columns, but are ultimately limited by the capacity of the two-dimensional surface onto which the sample is deposited. Typically, about 1 microL of chromatographic effluent plus 1 microL of matrix solution can be deposited and dried on a ca. 3 mm diameter spot. Deposition rates (spot dwell time) are determined by the chromatographic resolution and the flow rate. To overcome this limitation, a new three-dimensional MALDI sample plate has been developed using collimated-hole structures (CHS) with monolithic chromatography media filling the holes in the collimated-hole structures. These new plates retain all of the required features of conventional sample plates, commonly formed from stainless steel, but provide additional capacity for capturing and concentrating samples. Results are presented from reversed-phase separation of peptides on a 1 mm i.d. column operating at flow rate of 50 microL/min. Typically, 10 microL of effluent can be collected on a single spot, and sample and matrix dried on a 1 mm diameter spot, to yield about 30-fold enrichment of sample concentration in matrix crystals on the surface compared to the conventional plate. Sample loadings ranging from 1 fmol to 10 pmol/spot were investigated.

Off-line coupling of high-resolution capillary electrophoresis to MALDI-TOF and TOF/TOF MS.

High-resolution capillary electrophoresis has been coupled to MALDI-TOF and TOF/TOF MS through off-line vacuum deposition onto standard stainless steel MALDI targets. This off-line approach allowed the decoupling of the separation from the MS analysis, thus allowing each to be independently optimized in terms of time. Using BSA tryptic digest as a model sample, the deposited streaks, roughly 100-microm wide, were first analyzed in the MS mode, consuming only a fraction of the sample. After data analysis, segments of the deposited trace, containing unidentified peptides, as well as several species chosen for sequence confirmation, were reanalyzed in the MS/MS mode using MALDI-TOF/TOF MS. Additionally, it is shown that the shot-to-shot reproducibility of the vacuum-deposited trace (5% RSD) is 1 order of magnitude lower than that found for the standard dried droplet method. Moreover, a linear dependence of signal intensities (relative to an internal standard) over 3 orders of magnitude was found for a peptide sample with concentrations ranging from 1 to 1000 nM. This paper demonstrates the potential of off-line coupling of high-resolution separations to MALDI-MS and MALDI-MS/MS using vacuum deposition for the analysis of complex peptide mixtures from protein digests.

Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins.

Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.