The main task of the Mass Spectrometry Facility is to provide an effective and highly sensitive analytical chemistry tool to complete molecular biological and biochemical methods in biological and medical investigations. Our activities are concentrated on the analysis of biopolymers (e.g. peptides, proteins and oligonucleotides). We revise and develop continuously methods for the fast and sensitive analysis of these molecules. To accomplish this challenge there are the following instruments available:
Each LC-MS/MS system is on-line coupled to an LC Packings UltiMate HPLC with Famos autosampler and Switchos column switching unit. Both HPLCs run at 250 nl/min flow rate to enable a direct interfacing to the mass spectrometers.
Our computational capacity has been recently improved with an 8 CPU Linux cluster for Mascot in order to decrease database search time.
Bruker Reflex III MALDI-TOF MS
MALDI-TOF MS is an abbreviation for Matrix-Assisted Laser Desorption/Iionization Time of Flight mass spectrometry. With this technique only solid samples can be analyzed. The sample is co-crystallized with a so-called matrix on the sample slide and the desorption of the analyte molecules is carried out by laser shots. This instrument is furnished with a UV laser. There are special types of matrices for each application. The matrix compounds which are applied, absorb the laser light, evaporate and transfer also the sample molecules into the gas phase without the danger of thermal degradation. The sample compounds will be ionized in the gas phase and accelerated into the flight tube. The time needed to reach the detector situated at the end of the field free drift tube is characteristic and proportional with the mass of the ionic species. Hence with the exact measurement of the time of flight the mass of the ions can be calculated. Ions having the same molecular mass, but different initial conditions can decrease the accuracy of the measurement due to slightly different flight times. To overcome this difficulty this instrument is equipped with a so-called reflectron or ion mirror, to focus the ions, having the same molecular mass, into small "packages". With this option the resolution and mass accuracy can be substantially increased (reflectron modus). Linear modus is applied for the measurements of proteins up to several hundreds kDa. Reflectron modus is preferred for peptide mapping experiments or for small molecules.
Applied Biosystems QSTAR PULSARi LC-MS/MS System
Enzymatic peptides, or intact proteins are desalted and concentrated on a 300 µm ID reversed phase trapping column on the Switchos module and eluted onto the 75 µm ID analytical column of the same material at a flow rate of 250 nl/min. The column material is chosen according to the analyte molecules. The eluate passes the UV detector and enters the mass spectrometer without splitting. In case of static electrospray measurements, peptides or proteins are purified and concentrated on a special reversed phase material and eluted into a metal plated silica capillary in a small solvent volume. The very narrow opening of this nano–spray capillary allows low (< 100 nl/min) flow rates and enough time to carry out sequencing experiments for several peptides even with small (1-1.2 µl) sample volumes. First the complete peptide spectrum will be acquired and the charge states of the peptides will be determined. Doubly or triply charged ions are preferentially chosen for peptide sequencing. These ions are selected in the quadrupol mass filter (Q1). In case of LC-MS/MS measurements this happens in an automated manner - by data dependent acquisition. Selected ions collide with nitrogen atoms in the high pressure collision cell and the resulting fragments are separated in a second, time of flight analyzer, which is equipped with an ion mirror or reflectron. The fragments reach the detector after a given flight time which is proportional to their masses. From these spectra structural information can be deduced. Because of the high resolution (7000 FWHM at m/z 830 in LC/MS modus) and excellent mass accuracy (5ppm with internal, 50 ppm with external calibration) this instrument is used for the identification of proteins (LC-MS/MS), de novo sequencing (with static nanospray) and for the molecular mass measurements of intact proteins or oligonucleotides.
Enzymatic peptides are desalted and concentrated on a 300 µm ID reversed phase trapping column on the Switchos module and eluted onto the 75 µm ID analytical column of the same material at a flow rate of 250 nl/min. The column material is chosen according to the analyte molecules.The eluate passes the UV detector and enters the mass spectrometer without splitting. First the complete peptide spectrum is acquired, then generally up to 4 data dependent MS/MS experiments are carried out. The advantage of this linear ion trap mass spectrometer compared to the QSTAR is the substantially shorter duty cycle. This makes it ideal for the analysis of complex mixtures. Neutral loss scans for the detection of e.g. phosphorylated peptides are applied routinely embedded into data dependent measurements. This instrument is mostly applied for the identification of peptides and proteins and for the determination of post translational modifications.
©ECs June 2005