Maldi
 
Protocols
 
 
 
 
 
 
Introduction

Prior to mass spectroscopy proteins in a biological sample are usually separated by one-dimensional or two-dimensional gel electrophoresis and made visible by silver- or, better, (colloidal) Coomassie staining. Each protein on the gel can principally be identified by MALDI-TOF MS. The analysis involves a number of steps consecutively consisting of:
  • Spot-picking
  • In-gel digestion and peptides extraction
  • Application of the sample to the target plate
  • MALDI-TOF MS
  • Protein identification
Spot-picking

Protein spots can be cut from the gel using a scalpel. When large numbers of proteins are to be analyzed, it is more convenient to do this with a robot. An Ettan™ Spot Picker (Amersham Biosciences) is available at the Nijmegen Proteomics Facility (contact: L. Lambooy).

In-gel digestion and peptides extraction

In-gel digestion is done by the protocol described in [8]. The procedure is improved by the following minor modifications:
  • After the standard washing/dehydration steps, each gel piece (a cylinder of 1.7 mm3) is re-swelled on ice with 5 ml 12.5 ng/ml trypsin in 25 mM ammonium bicarbonate for 1 hour.
  • Hereafter, excess trypsin mixture is withdrawn and the gel piece is covered with 5 ml of 25 mM ammonium bicarbonate containing 0.1% n-Octyl-Pyranoglucoside. The sample is incubated for 14 hours at 37°C to allow complete digestion.
  • To extract the peptides, 1 ml 0.5% TFA in 10% acetonitrile is added to the sample. The mixture is incubated for 1 hour at room temperature and subsequently sonicated for 2 minutes.
  • A small portion of the extracted fluid (usually 0.2 ml) is directly taken for MALDI-TOF analysis, i.e. without vacuum-drying.
Application of the sample to the target plate
  • Before MS analysis the extracted peptide mixture is spotted to a metal target plate together with a matrix solution. Matrix solution contains 20 mg/ml 4-hydroxy-a-cyanocinnamic acid in 50% acetonitrile/ 0.05% TFA.
  • To the target plates used here, 384 samples can be applied. The plates should be thoroughly cleaned. This is done by consecutively washing with ethanol, isopropanol, and methanol. To ensure proper cleaning, each washing step is evaluated under a microscope. If final washing with methanol is not sufficient, an additional sonication of the target plate in methanol is performed.
  • By routine, a 0.5 ml of a protein mix is analyzed for external calibration of the mass spectrometer. The calibration mix contains 1.26mM bradikinin, 1.26mM P14R, 0.19mM angiotensin and 0.95mM ACTH in matrix solution. Series of eight samples are spotted around one external calibration mixture.
  • A critical step in MALDI-TOF MS is the application of the peptide sample. In literature, the sample is usually pre-mixed with the matrix, spotted and the mixture is air-dried. The pre-mixing, however, may result in lower detector signals. Therefore, we prefer a two-step application. First, 0.2 ml of the peptides extract is directly spotted to the target plate, directly followed by the spotting with 0.2 ml matrix solution. The mixture is allowed to air-dry. In our hands, the two-step procedure results in a more homogeneous distribution of the protein-matrix crystals and in higher detector signals.

MALDI-TOF MS

After protein spotting, the sample plate is transferred into the mass spectrometer (Bruker Biflex III MALDI-TOF MS; Bruker Daltonics). The machine is schematically shown in the Figure below, together with recommended experimental settings.

To get the best results with respect to accuracy, resolution and sensitivity, the spectrometer can be operated in different modes and experimental settings. The settings depend on the nature of the sample and the finding of the optimal conditions may be a matter of trial and error. In our analysis of the Methanothermobacter thermautotrophicus proteome we used the following (see also: Figure 4):

  • pulsed-ion extraction (PIE)
  • Reflectron mode.
  • High-voltage- and low-molecular-weight-suppression. By this setting, low-molecular-weight matrix adducts are deflected, resulting in the improved signal-to-noise ratio for peptides <750 Da.
  • Maximum acceleration voltage of the first electrode: VIS/1= 19 kV. The value is determined as follows: optimal reflection by the reflectron is achieved at 105% of the applied acceleration voltage, which is limited at 20 kV. Thus: VIS/1= 20 kV/1.05 =19 kV.
  • Maximum acceleration voltage of the second electrode: VIS/2= 9.5 kV, i.e 50% of VIS/1. To obtain a real-time VIS/2= 9.5 kV, the electrode should be set at 15.6 kV.
  • Detector voltage: 1.9-2.0 kV (setting recommended by the manufacturer: 1.7 kV). The higher detector voltage enhances the peak resolution and signal-to-noise ratio. This is not necessary for high protein concentrations and, in fact, not very advisable. The detector life time is reduced and it may lead to a stronger detection of ghost peaks (false peaks caused by impurities or matrix adducts) and, eventually, to detector saturation by strong intensity peaks. In its turn, detector saturation may result in the loss of weak peptide signals, which escape detection.
  • Peak shapes are empirically optimized by changing the lens kV setting (recommended: 9.1 kV).
  • Laser intensity detenuation: 50-55. Higher laser intensity (>55) may cause peptide fragmentation, whereas lower intensity (<50) results in lower detection signals.
Figure 4 - Maldi parameters (click to enlarge)
The MALDI-TOF mass spectrum is generated by mathematically averaging the spectra obtained from multiple N2-laser shots. In practice, 120-180 shots should be sufficient. The exact position, where the laser hits the spotted sample is seen on a monitor connected to a microscope which is focussed on the sample. Inspection through the microscope also gives an impression of the quality and distribution of the protein-matrix crystals. It is advisable to collect spectra from different positions within the sample.
 
Protein identification
  • All (relevant) peaks seen on the MALDITOF MS spectrum are manually annotated as their mono-isotopic peptide peaks ( [M+H]1+ ). The peaks with their respective m/z values are stored in a peak search list using the X-TOF program (Bruker Daltonics).
  • The search list representing the protein peptide mass fingerprint is compared to genomic databases. This is done by the MASCOT search algorithm (MATRIX SCIENCE). MASCOT search uses a peptide mass mapping search algorithm, which compares experimentally obtained m/z values with theoretically calculated m/z values of tryptic peptides of proteins from a genomic open reading frame database.
  • Searching is done under the conditions of a preset peptide mass error and a preset number of trypsin missed-cleavages. The preset parameters signify the statistical confidence of the score. For instance, when the MASCOT search is done allowing a 0.2-0.3 Da peptide mass error and one missed cleavage by trypsin, the chance of a false, coincidental protein identification is less then two times the standard deviation (P<0.05). In addition, the statistical significance of a hit is expressed as its ‘MOWSE score’: the higher this number, the higher is the reliability of the hit.
  • Note: It can be very useful to check the theoretical protein pI’s and/or molecular masses of the positive hits, i.e. as given in the genomic database, against the experimentally determined values obtained by two dimensional electrophoresis. The matching of the experimental and theoretical values, obviously, improves the reliability of the score.