Dirk Chelius, Terry Zhang, and Ken Miller;
Thermo Electron Corporation, San Jose, CA
Key Words Sensitivity Proteomics Finnigan LCQ Deca XP Plus PepFinder Kit Finnigan Surveyor HPLC LC/MS/MS
Detection limits of mass spectrometers have been pushed downward to levels no one would have thought possible only a decade ago. Sensitivity gains are driven by instrument hardware improvements and sophisticated methods of sample introduction to the mass analyzer. Nanoflow ESI is a very popular method in proteomics to achieve maximum sensitivity and separation of peptides. Peptides are separated on a capillary reversed-phase column (75 μm internal diameter [ID]), which is placed directly in front of the mass spectrometer to limit peak broadening after the column. For many reasons, use of these small ID columns can be complex. Engineering an optimal solution to these practical issues can be very time consuming. The optimal flow rate for a 75 μm ID column is between 100 and 200 nL/min, often requiring flow splitting. At such low flow rates, loading of a sample onto the column can take a long time: at a flow rate of 100 nL/min it takes 200 minutes to elute a 20 μL sample loop. Researchers have attempted to overcome this problem by loading the sample onto the column during the packing of the column, but this method is very time consuming and each column can only be used one time.Alternative approaches are the loading of the samples at a higher flow rate or the use of a peptide trap. The peptide trap has several advantages compared to other loading techniques; peptides can be loaded at a higher flow rate, desalted and washed. This combination allows high sample throughput and maximal sensitivity of detection. This plumbing configuration is the basis of the PepFinder Kit. Designed for easy installation and maximum productivity, all PepFinder Kit componen ts come pre-assembled, minimizing time required for system setup and optimization. Materials, plumbing configurations, and connection of flowpath components are all engineered for optimum performance.
The goal of this study was to evaluate the sensitivity, reproducibility and robustness of the PepFinder Kit, using the Finnigan LCQ Deca XP Plus ion trap mass spectrometer. The ability of the Finnigan Surveyor HPLC system, used with an optimized splitting system, to deliver consistent flow at flowrates rates of 100200 nL/min was also evaluated.
Protein standards human serum albumin, human IgG, human transferrin, human hemoglobin, human macroglobulin, and horse myoglobin were purchased from Sigma-Aldrich, St. Louis, MO, USA.
Reduction, alkylation and digestion
One mg of each lyophilized protein standard was reconstituted separately in 1 mL of ammonium bicarbonate buffer (100 mM, pH 8.5) and 3 μL DTT (1 M, Sigma- Aldrich, St. Louis, MO, USA). The mixture was incubated for 30 minutes at 37C. To alkylate the protein, 7 μL of iodoacetic acid (1 M in 1 M KOH, Sigma-Aldrich, St. Louis, MO, USA) was added and the mixture was incubated for an additional 30 minutes at room temperature in the dark. Thirteen μL DTT (1 M) was added to quench the iodoacetic acid. The reduced and alkylated proteins were digested by adding 20 μL modified trypsin (0.5 mg/mL, Promega, Madison, WI, USA). The mixture was incubated for 6 hours at 37C, then an additional 20 μL trypsin (0.5 mg/mL) was added and incubation was continued for 16 hours at 37C.
The digested samples were analyzed using a fully automated nanoflow LC/MS/MS system, configured with a PepFinder Kit (Figure 1). Aliquots of 10 μL were placed in wells of a 96-well plate (Nalg e Nunc International, Rochester, NY, USA). The plate was sealed with plastic film to minimize evaporation and inserted into the autosampler of a Finnigan Surveyor HPLC system (Thermo Electron, San Jose, CA, USA), where it was kept at 4C while waiting for analysis. The injected peptides were first loaded onto a reversed-phase poly(styrene-divinylbenzene) peptide trap (Michrom BioResources, Auburn, CA, USA) with a flow rate of 10 μL/min for 3 minutes (Figure 1). The peptides were eluted from the trap and separated on a reversed-phase capillary column (PicoFritTM; 5 μm BioBasic C18 [Thermo Electron], 300 pore size; 75 μm 10 cm; tip 15 μm, New Objective, Woburn, MA, USA) with a 30-min linear gradient of 060% acetonitrile in 0.1% formic acid/water at a flow rate of approximately 0.1 μL/min after split. The HPLC was directly coupled to a Finnigan LCQ Deca XP Plus ion trap mass spectrometer equipped with a nanospray ionization source. The spray voltage was 2.0 kV and the capillary temperature was 150C. The ion-trap collisional fragmentation spectra were obtained using collision energies of 35%. Each full-scan mass spectrum was followed by three Data DependentTM MS/MS spectra of the three most intense peaks. The Dynamic ExclusionTM feature was enabled (Repeat Counts: 2, Repeat Duration: 0.2 minutes, Exclusion Duration: 5 minutes and Exclusion Mass width: 2 Da).
Peptides and proteins were identified automatically by the computer program BioWorksTM version 3.1 (Thermo Electron, San Jose, CA, USA) which correlated the experimental tandem mass spectra against theoretical tandem mass spectra using the SEQUEST algorithm. The protein database used for this analysis was obtained from the National Center for Biotechnology Information (NCBI). Peptide identification was evaluated using the Xcorr vs. Charge State fil ter which accepts only peptides with Xcorr values of 1.5, 2.0, and 2.5 and higher for singly, doubly, or triply charged precursor ions.
Results and Discussion
The tryptic digest of horse myoglobin was analyzed by nanospray liquid chromatography as described in the Experimental section. The LC/MS system was run under standard conditions one would use for typical protein identification in a complex mixture of proteins and not optimized for the detection of horse myoglobin digest. The mass range for full mass spectrum was 400 Da to 1500 Da and each full mass spectrum was followed by three MS/MS scans of the most intense ions. Dynamic Exclusion was enabled. Horse myoglobin could be identified in amounts as low as 500 amol. Figure 3 shows the BioWorks results and Figure 4 shows one of two tandem mass spectra obtained for this low concentration. Both tandem mass spectra contained sufficient fragment ions for positive identification by the SEQUEST algorithm. The results were confirmed several times after rigorous washing of the LC (peptide trapPicoFrit column) system.
The Finnigan Surveyor HPLC system, used with an optimized flow splitting system, generated very reproducible chromatograms at flow rates of 100 nL/min. Two different amounts (250 fmol and 500 fmol) of a tryptic myoglobin digest were analyzed three time each and the elution time of each peptide differs by less than 10 seconds from run to run, which is less than 0.3% of the elution time (Figure 5).
Overall system reproducibility was further evaluated by analyzing a mixture of all five standard protein digests at various concentrations ranging from 10 fmols to 6 pmols. Forty replicate analyses were performed. All five proteins could be identified in each of the 40 analyses, using BioWorks software. Peptide coverage for each protein was very similar between all runs, demonstrating excellent reproducibility of LC/MS with t he Finnigan Surveyor, Finnigan LCQ Deca XP Plus, and PepFinder Kit (Table 1).
The PepFinder Kit eliminates much of the complexity associated with nanospray LC/MS, providing easy installation and robust system operation. These benefits come with no sacrifice of system sensitivity or performance. The identification of sub-fmol proteins can easily be achieved using the Finnigan LCQ Deca XP Plus ion trap mass spectrometer in combination with the PepFinder Kit.