Dr. Christie Hunter: Welcome, and thanks for joining us today. My name is Christie Hunter, and I'm the Director of Pharma and Proteomics Applications here at ABSCIEX. Today, I would like to talk about the new QTRAP 4500 System. I'll tell you a little bit about the technology and then discuss how this exciting new platform can be used for protein peptide quantification.

First, I would like to show you the ion rail [sp] of the QTRAP 4500 system and discuss the specific key instrument features. The first think you notice is that system has a curved ion rail. It is based on the instrument configuration of the QTRAP 5500 system, so the instrument has a nice small footprint.

We start from the front of the instrument at the atmosphere pressure interface. Here, the orifice is the same size as the 4000 QTRAP. Behind the orifice is a QJet ion guide. The QJet is extremely efficient at capturing the ions coming through the orifice and quickly focusing them into the QZERO [sp] quadrupole. This is the same technology as the QTRAP 5500 system and that was pioneered the APLAD [sp] 5000 system.

After QZERO, we have our first mass analyzing quadrupole. Q1 and Q3 quadrupoles operate at a lower RF frequency than on the QTRAP 5500, which enables a mass range of up to 2,000 M over Zed to be obtained in both quadrupole and ion trap modes.

Next, the ions will transit the curved Q2 collision cell, which has higher potential gradient across the collision cell. This enables shorter ion transit times and more MRMs per unit time. The ions then enter the linear accelerator ion trip. This was another big innovation that was first enabled on the QTRAP 5500 system, bringing LINACK [sp] technology to the Q3 linear ion trap.

This provides very large increases in sensitivity. Ions are more efficiently captured, cooled and extracted, providing a big benefit in full scan sensitivity and then also in the MRM cubed workflow.

Finally, the ions arrive at the accurate pulse counting detector, which provides very high reproducibility and accuracy.

The instrument also has the EQ Electronics of the QTRAP 5500 system, which provides fast quad scanning, fast linear ion trap scanning, fast priority [sp] switching and much shorter field time.

So, let's talk a little bit about the basic performance of the QTRAP 4500 system. In terms of the MRM sensitivity for peptide and protein quantification, the QTRAP 4500 performance is typically a little better than the 4000 QTRAP. So, in terms of sensitivity, it will fit somewhere between the 4000 and 5500 levels in terms of peptide MRM quantification. This slide shows a couple of examples, a large peptide insulin and a tryptic peptide glu-fib, and here we see about a 3X gain in signal.

One of the unique capabilities of the QTRAP technology is--that really differentiates from using just a triple quadrupole platform is the ability to perform high sensitivity full scans in the linear ion trip. Shown here is the sensitivity difference between doing a full scan using a quadrupole on the QTRAP 4500 system versus a full scan using the linear ion trap mode. There's roughly about a 30X increase in sensitivity at about a 10X faster scan rate. This allows full scan MS or MS/MS data to be collected in an LC timeframe enabling qualitative data to be collected right along with the quantitative data.

The other point to make regarding ion trap sensitivity is that the LIP mode on a QTRAP 4500 system is almost as sensitive as a QTRAP 5500 system. This is because the QTRAP 4500 system is using the linear accelerator trap technology that was originally developed on the 5500 system. Here we see about a full X lower sensitivity in ion trap mode on the QTRAP 4500 system. However, the mass range on the QTRAP 4500 system is higher. In both quadrupole mode and linear ion trap mode, you can scan to an upper mass range of 2000 M over Zed.

Shown here is insulin acquired in MRM mode on fragment ions across the mass range on the top. The bottom is a full scan linear ion trap MS/MS that was triggered from the MRM scan. This highlights one of the unique capabilities of the QTRAP technology, the ability to combine targeted quadrupole scan modes with the high sensitivity qualitative ion trap modes. Here is another example of combining scan function.

For functional relation analysis serum [sp] and premium [sp] phosphor peptides can be specifically targeted by providing a neutral OP [sp] scan for the [unintelligible] phosphate group. When signal is detected the neutral OP scan, it can trigger a high resolution enhanced resolution scan for more accurate mass determination and charge date determination. This one triggers a full scan MS/MS spectrum that can be used to ID the peptide and localize the photophosphorylation.

The Midas [sp] workflow is another example of the hybrid quadrupole linear ion trap workflow. Typically, MRM scans are used for quantitation of analytes and peptides in complex matrices. But, they can also be used to target the detection of specific peptides. Here we can use MRMs to target peptides from a specific protein of interest by designing MRMs in silico. These are then used as survey scans, and when signal is detected for one of the MRMs, it triggers acquisition of a full scan MS/MS to identify the peptides.

In this example, we have detected the protein glutaredoxin 2 from e-coli, but only one observed confident peptide IB. MRMs were designed for the other peptides to the protein and then targeted using the Midas workflow in the digestive e-coli matrix.

Shown here is the result for one of the additional detected peptides from the protein, peptide SAF [sp]. Five MRMs were monitored for the peptide and full scan MS/MS was triggered, and this MS/MS was then subjected to a database search, which identified the extra glutaredoxin peptide. In this example, three e-coli proteins of interest were identified with a single peptide identification. The proteins are glutaredoxin 2, outer membrane protein SLP and purine nucleoside phosphorylase.

Using the Midas workflow, additional peptides to each protein were targeted to increase the number of peptides per protein and increase the confidence of detection. In each case, an additional three to four peptides were found with increase the sequence coverage obtained on each protein to over 25 percent. Now, with the data at hand, a high quality MRM assay using multiple peptides per protein can be developed to quantify each of the proteins.

After you've developed your quantitative MRM assay, you could build highly multiplexed MRM assays using the scheduled MRM algorithm. When we know the elution time of the peptide, we can use this time information to intelligently schedule the acquisition of the MRM for that peptide. Because peptides are alluding at various times across the LC gradient, each MRM is monitored only across its expected elution time. This decreases the number of concurrent MRMs at any one time, allowing the user to maintain both the cycle time and the dwell time. This increases the effective duty cycle.

The workflow is extremely easy to set up, and with the new Analyst 1.6 software, one can schedule up to 4,000 MRMs at a single run on both the QTRAP 4500 and 5500 systems.

To explore the feasibility of running 4,000 scheduled MRMs in a single workflow, an experiment was designed in which we monitored 25 peptides in a complex e-coli matrix. We ran 200 micron ID chips in travelute [sp] mode at a flow rate of one microliter per minute using the exigent nano LC Ultra with the Chip LC Nanoflex.

To add a higher level of multi flexing, random MRMs were generated at random retention times and added to the assay to increase the number of MRMs. The reproducibility of original 25 peptide MRMs were then monitored for their raw peak area of reproducibility across ten replicates. The data is displayed as a cumulative frequency plat where the CV is plotted on the X axis and the percent peptides is on the Y. So, using 10 percent CV as our reference point, you can see that even with 2,000 or 4,000 MRMs monitored in a single blend [sp], 65 to 75 percent of the data has CVs less than 10 percent.

The cytochrome P450 protein super family of mono oxygenases is the major phase one enzyme system responsible for metabolism of most drugs and other foreign chemicals. In higher eukaryotes, there are over 70 families of P450s involved in drug metabolism comprising of over 200 different isoforms [sp] with different substrate specificity.

Members of the same family of SIT [sp] proteins designated by the same number of share greater than 40 percent sequence identity. When you consider proteins in the same subfamily sharing both a number and a letter, the identity is over 55 percent.

In this example, the SIT 450 protein 3A4 and 3A5 share over 84 percent sequence identity. So, when designing quantitative MRM assays to proteins of families such as this, having peptides that uniquely identify the target protein can be very challenging. The peptide selection will be limited and the peptides that you can use will not always be the most [unintelligible] or the best behaved peptide.

This means that they could be more prone to interferences when monitoring and in complex matrices. So, I'd like to discuss the MRM cubed workflow on the QTRAP 4500 system that can be used for quantitative peptide assays when more selectivity is required.

When performing an MRM cubed experiment in the QTRAP system, the first precursor ion is selected in Q1, fragmented in Q2 and all product ions are trapped in the linear ion trap. Then, the second precursor ion is isolated and fragmented again in the linear ion trap.

Finally, the second generation product ions are then scanned towards the detector. This scan is collected over and over again in a looped fashion and then extracted ion chromatograms are generated post acquisition for quantitation.

With this extra isolation and fragmentation step, MRM cubed is more selective than MRM mode. It can be useful for quantitation in cases of high noise and background interferences. The first peptide that we targeted was the BPI peptide from cytochrome P450 3A5. Shown on top is the full scan MS/MS factor that was collected on the light peptide.

Two dominant Y ions were observed. The Y5 ion was then selected and further fragmented in the ion trap to produce the MS/MS/MS spectrum shown on the bottom. A very nice fragmentation pattern was obtained with multiple fragment ions that uniquely identify the target peptide.

Six secondary product ions were selected and XICs [sp] were generated, and these were then summed together to produce the MRM cubed data. Integration of the pecaria [sp] of the MRM cubed is what is used for quantitation.

The unique peptides for the cytochrome P450 3A5 were dosed into human liver microsomes to develop the quantitative assay. Light peptides were dosed in at a fixed amount as an internal standard, and the heavy peptides were used to generate a standard [sp] concentration curve in order to determine the LLLQs [sp] for the peptide. Both MRM transitions and MRM cubed data was collected on all the points in the curve.

On top are three MRMs to the DTI peptide. You can see that two out of three of the MRMs show significant interferences in the MRM signal. The data is actually shown at 4.8 centimoles on column, which is about 4X higher than the LLLQ, in order to visualize the interferences in the MRM data.

However, when the quantitation is done on the MRM cubed data, shown on the bottom, a much cleaner peak is observed.

The standard concentration curve was linear across the concentration range explored from the LLLQ of 1.2 centimoles on column to 805 centimoles on column. Higher points were not analyzed. The data at the bottom is the MRM cube signal at the LLLQ where the CV was 4 percent and the accuracy was 83 percent.

All the data processing was done in Multi Point software. The second peptide monitored was the SLG peptide from 3A5. Again, both MRM and MRM cubed assays were run on the standard concentration curve. Data shown here is the MRM and MRM cubed data at 4.8 centimoles on column. And again, two out of three of the MRM transitions showed significant interferences that impacted the LLLQ values, whereas the MRM cubed data shown on the bottom right also provided a clean peak for quantitation.

Similar LLLQ for MRM cubed were achieved on this peptide, 1.2 centimoles on column. The MRM transitions provided LLLQs of 4.8 centimoles on column, about four times worse than the MRM cubed data.

In summary, quantitation of peptides and proteins using MRM assays remains a workhorse for targeted peptide quantitation in complex matrices. However, sometimes, additional selectivity is required.

The MRM cubed workflow on the QTRAP 4500 system can be used to provide additional selectivity when facing tough quantitative challenges.

In conclusion, the new QTRAP 4500 system is an exciting new hybrid triple quadrupole linear ion trap system that enables powerful and unique workflows for qualitative and quantitative proteomics. Because of the unique hybrid nature of the ion rail powerful workflow such as the Midas workflow and the [unintelligible] scan or neutral law [sp] scanning can be used to target specific peptides or classes of peptides. Coupled with the very high sensitivity linear ion trap for folk [sp] scan analysis, the identities of these can then be confirmed.

Moving into targeted quantitation, the scheduled MRM algorithm provides an easy to use robust method to maintain the highest quantitative quality while maximizing throughput. Assays up to 4,000 MRMs in a single run can now be performed.

Finally, while MRM analysis remains the main method for most assays, the increased selectivity of MRM cubed allows for the elimination of chromatographic interference resulting in superior analytical performance compared to traditional MRMs when additional selectivity was required. Finally, I would like to thank my colleagues at AB SCIEX for their work on the various projects, and I'd like to thank you for your attention.