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Gel electrophoresis
An integrated package (BIO-RAD PROTEAN IEF cell and vertical slab electrophoresis cells) using pre-cast gels allows simultaneous development of up to twelve 11 cm2 (CriterionTM) or 17 cm2 (DodecaTM) gels. Immobilized pH gradient (IPG) strips are available in a variety of pH ranges (3-6, 5-8, 7-10 and 3-10) to optimize separations. Pre-loaded methods for the IEF cell, which has a 10,000 V capacity, allow easy start-up for the novice.
Gels can be stained with Coomassie blue, silver stain, or Sypro Ruby. The latter is a fluorescent stain with sensitivity equal to that of silver stain, with the advantage of a quantitative response over three orders of magnitude. In addition, this stain does not interfere with subsequent mass spectrometric characterization of proteins and peptides.
ProteomeWorks Spot Cutter: Spot-cutting
FX Molecular Imager & GS-800 Densitometer: Imaging
A ProteomeWorks spot cutter system with its associated PDQuest software is used for automated imaging, location, analysis, excision and transfer of spots into 96-well plates prior to in-gel digestion. Six or more different gels can be compared to a master gel (a limit of ca. 5000 spots), and spots chosen for excision, digestion and identification based either on qualitative or quantitative criteria. Both visible (GS-800 Calibrated Densitometer) and UV (FX Molecular Imager) imagers are available, allowing use of a variety of staining procedures.
Quantitative and qualitative analyses are possible, as well as multiple cuts of large spots, and pooling of the same low-abundance spots from multiple gels into a single microtiter plate well. Cutting of spots can be done directly using analysis sets generated using criteria determined by the user. Spot cut lists can be exported to spreadsheets or other data bases to facilitate data tracking.
MassPREP Robot: Proteolytic digestion
After spot excision, the 96-well plates to which excised spots are transferred are moved to a Micromass MassPREPTM robot for automated reduction, alkylation, proteolytic digestion and peptide extraction. Approximately 10% of a given digest is automatically spotted for MALDI analysis, with sufficient sample left for subsequent LC-MS analysis if necessary. The robot is easily programmed to accommodate additional steps, such as ZipTip purification, multi-step chemical modifications of specific residues, or sequential addition of two or more different proteolytic enzymes. Programs are also available for digestion of LC fractions.
Mass Spectrometers
Micromass M@LDI -TOF HT
The Micromass M@LDI -TOF HT (reflectron mode only) is used for automated analysis of peptides and protein digests of 200-1000 samples per day.
Agilent 1100 LC/MSD Ion Trap
An Agilent 1100 LC/MSD ion trap mass spectrometer with an autosampler configured to 96-well plates is available for LC-MS and LC-MS2 analyses. It is coupled with a capillary LC system that reliably maintains flow rates of 150 nL/min for nanospray analyses. Samples not successfully identified by MALDI MS are analyzed using LC-MS2 on either the ion trap or the Q-TOF, or the atmospheric pressure ionization (API) MALDI source for the ion trap. The ion trap can also perform MSn analyses, providing detailed structural information for many molecules.
The atmospheric pressure ionization MALDI source for the ion trap accommodates 100-spot plates. A single spot can be used for collecting 30 high quality MS2 spectra in ca. 7 minutes, allowing definitive identification of most digested proteins not identified from Micromass M@LDI spectra.
Applied Biosystems QStar XL
Two hybrid quadrupole time-of-flight Q-TOF instruments (QStar XL, Sciex/Applied Biosystems) can be used for high resolution/high mass accuracy peptide mapping, with both electrospray ionization (ESI) and MALDI sources available for sample introduction. Mass ranges up to 6,000 for MS2 and 40,000 for MS provide a flexible platform for characterization of most proteins and peptides. Samples for ESI analysis can be introduced either by direct infusion or on-line LC.
Applied Biosystems Voyager-DE STR
Two MALDI-TOF instruments (Voyager-DE STR, Applied Biosystems), with linear and reflectron capabilities, provide automated data acquisition and processing for purified or complex mixtures of peptides (low- to mid-femtomole levels) and proteins (low picomole sensitivity, mass range > 100 kDa). MALDI is a robust technique that tolerates moderate levels of buffers and salts. This characteristic and fast acquisition times (5-60 seconds) make MALDI ideal for screening extracts during lengthy purification procedures.
IonSpec Ultima 7T FTICR
For samples requiring very high mass resolution (50-70,000) and mass accuracy (+/- 0.02 millimass units), a fourier transform ion cyclotron resonance (FTICR) instrument (IonSpec Ultima 7T) is available.