Mass spectrometry has become an essential technique in the field of proteomics as peptide mass fingerprinting and sequencing are now an important tool to identify unknown proteins. The purchase of the Ultraflex MALDI TOF/TOF instrument from Bruker Daltonics was to procure an instrument for accurate mass determination of different biological and chemical samples using only miniscule amounts of the material. Essentially, the Bruker Daltonics Ultraflex consists of two Time-of-Flight channels. The first separates the ions generated by laser beam on the basis of their molecular weights giving a mass fingerprint. The second TOF resolves the fragmented species generated by a collision chamber, which is present between the two TOFs.
We have been able to fragment peptides (generated from tryptic digests of proteins) of upto 3000 Da and the fragmentation of higher mass peptides is being standardized. We also carry out routine mass determination of proteins with molecular weights as high as 80 kDa. The accuracy of the instrument is about 0.1 Da in the low molecular weight range (upto 8 kDa) and drifts to about 5-20 Da when the mass range hits the 75 kDa range, depending on the laser power employed. Theoretically, the upper limit of the mass detection range is set at infinity. However, we have been having problems getting large molecular weight proteins (about 90-120 kDa) to ionize well.
The advantage of the instrument, however, is in its sample requirements. Even for fragmentation (LIFT) experiments, the maximum sample required has not exceeded the femtomolar range. The best part is that the same sample can be used for peptide mass fingerprinting, as well as, MS/MS. Only 1 ul of sample is needed per spot, which greatly reduces the demand of purified sample. Spots obtained on SDS gels can easily be cut out and processed for trypsin digestion and spotted on the MALDI target plate to obtain a spectrum. The instrument does not require the sample to be in its pure form, as the protein ionization is not affected by the presence of other protein/peptide molecules. Additionally, MALDI does not require the protein to be in a soluble form. Even protein aggregates and precipitated protein can be easily employed for MALDI analysis. The instrument also offers a wide range of solvents in which the sample can be supplied, ranging from buffers in water to highly viscous solvents like DMSO and DMF. Even strong acids like HCl and TFA can be used for sample preparation. Samples that are difficult to ionize can be detected by enhancing their ionization using volatile salts. Screening across a large number of conditions is also possible, as the plate offers the analysis of upto 384 samples before it can be washed for re-use.
The sensitivity of the instrument is remarkable, as we have obtained an excellent isotopic distribution of peptides and proteins, going upto the M+7 species for a 1.5 kDa peptide. This is astonishing because, in a small peptide, the probability that the same peptide molecule has 7 heavy isotopes of nitrogen and/or carbon is exceedingly low. Additionally, laser-induced di- and trimerization of proteins has also been noted. The instrument comes with its own software and a well-detailed manual on its use. For people comfortable with using the modern software supplied with most of the mass spectrometers available in the market, the Bruker software might seem complex and hard to understand. However, the software can be mastered in a matter of a few days and should be no problem, given the utility of the instrument. Flex analysis, a software package, from Bruker is generally used for the analysis of the spectrum and the assignment of the peaks.
Molecular Biophysics Unit
Indian Institute of Science
Bruker Daltonics Ultraflex MALDI TOF/TOF Mass Spectrometer
Machine is easy to handle and comes with 3 sets of spotting plates including patented Anchor Chip to deliver high sensitivity with minimal sample requirements.
Users might find it difficult to handle the software, but with a little training it can be mastered in no time.
The Bottom Line
A must-have for those who want to carry out routine proteomics work such protein and peptide characterization, identification of unusual sites on modification on peptides and proteins.