Dr. Christie Hunter: Hi. My name is Christie Hunter, and I would like to talk to you about our newest MS system, the TripleTOF 4600 system

This is an extremely exciting new platform for proteomic applications because of its ability to perform both qualitative and quantitative workflows with high performance and speed on a single instrument in a single workflo

The TripleTOF 4600 system is an extension of the TripleTOF family, building on the proven robustness and powerful workflows of the original TripleTOF 5600 system to provide systems with the highest productivity for proteomics research labs

The 4600 system has the same high-acquisition rates as the original system, collecting high-resolution MS and MS/MS spectra, but now on a value-priced platform

I'd like to first remind everyone about the hardware configuration of the TripleTOF 4600 system and discuss the key elements

Starting at the front end of the instrument, you can see it is very similar to the front end of our high-end Triple Quad instrument

The ions from the source first transit through the larger-diameter orifice into the QJet region

The QJet provides increased sensitivity and robustness through improved ion containment

This quadrupole operates at higher pressure, providing better collisional focusing and improved ion transmission

The ions are further cooled and focused in the Q0 region of the instrument and then are passed into the first mass-analyzing quadrupole

The collision cell is a high-pressure LINAC collision cell which accelerates ions through and provides fast transit time for fast MS/MS and multiplexed analysis

True collision-induced fragmentation provides reliable, information-rich spectra

The Accelerator TOF Analyzer is designed to deliver the speed and sensitivity needed for high-quality targeted quantitation but also deliver the high-resolution and mass accuracy of MS and MS/MS data for qualitative applications

Continuing through the ion rail, the ion compression optics behind the collision cell direct the beam into the accelerator, which is operating at 30 kilohertz, which provides high-duty cycle and instrument dynamic range.

The acceleration voltage of the TOF region is 10 kilovolts, which provides high sensitivity across the mass range

The mirror is a two-stage reflectron which compensates for energy dispersion and maximizes resolution

Throughout, high-transparency grids are used to ensure highest transmission and minimal ion scattering to maintain resolution

Finally, there is a 40 gigahertz multichannel TDC to provide the highest sampling speeds and maintain high resolution across the mass range, even at low M-over-Z region of the spectrum

So, putting it all together, the TripleTOF 4600 system is a powerful new QqTOF system that delivers all the qualitative analysis power of a high-resolution accurate mass analyzer but, at the same time, provides workflows for doing targeted quantitation workflows with triple-quadrupole-like data quality

So, here is a summary of the performance attributes of this new system. Sensitivity is one of the big distinguishing features of the TripleTOF system, both in MS and MS/MS modes

It is this sensitivity that allows us to achieve the extremely high-acquisition rates on this instrument

In information-dependent acquisition mode, MS/MS spectra can be acquired at up to 50 MS/MS in a second, maintaining high resolution even at these fastest rates

The resolution on this instrument is greater than 25,000 resolution in both TOF MS and MS/MS modes. This ensures good mass accuracy. We typically see less than 2 ppm RMS mass accuracy when identifying peptides from complex proteomes

And the dynamic range we typically see is about three to four orders linear dynamic range

To leverage the speed and specificity of this new MS platform, high-quality separations are critical

This is why we partnered with Eksigent technologies so we could combine the highest-performance nanoLC systems with our powerful MS system

The Eksigent nanoLC-Ultra system is a splitless nanoLC system that provides extremely precise and reproducible gradients

This is key to perform fast, high-quality separations with the run-to-run reproducibility that we need for proteomic and biomarker applications

The Eksigent cHiPLC-nanoflex system is a unique platform for chip-based LC separations

Eksigent has developed a very unique edge connection technology which enables very low dead volume microfluidic connections to be made easily and reproducibly

Therefore, the platform is very modular, enabling many different workflows to be performed on a single system

The microfabrication used in the production of the chips produces cylindrical column channels, which of course produces ideal chromatography and enables production of highly reproducible columns

The column-to-column reproducibility we see is less than 2 percent RSD, which is critical for our large biomarker discovery and verification studies where we have many samples to analyze and we need good reproducibly

Having the ability to perform both high-speed qualitative and high-sensitivity quantitative analysis on a single MS platform opens up a lot of possibilities for powerful workflows

We have focused on the delivery of solutions across the qualitative to quantitative continuum, from global exploration of a proteome sample to high-targeted high-resolution quantitation of proteins

Powerful software applications that enable easy data analysis are, of course, equally important for getting the most out of our MS data, and we've developed software solutions that cover this broad range of applications

First, I'd like to show you some data form non-targeted qualitative analysis and then finish with some workflows for high-resolution quantitation

As I mentioned before, the high MS/MS acquisition rates possible on the TripleTOF 4600 system really set this system apart for global discovery workflows

In this experiment, we ran a digested E. coli cell lysate, loading one microgram on column using a 60-minute gradient

The speed is illustrated here. The top pane shows how complex the total ion chromatogram is and how peptide rich the sample is

The bottom pane shows a computation of the number of MS/MS performed in each second across the gradient

The instrument automatically adjusts the acquisition rate such that more MS/MS acquisitions are performed in the peptide-rich region of the chromatogram, and less spectrum is acquired when fewer peptides are eluting

In this experiment, the maximum number of peptide per cycle was set to 20, and you can see that this maximum was hit across most of the sample

High-resolution TOF MS data was collected on all precursors, and of course, the high-resolution data translates into high-mass-accuracy data

Shown is an example of TOF MS and a zoom-in on one of the peptide precursors

The resolution of this peptide is seen to be 29,000 revolutions

Below is a summary of a mass-accuracy evaluation on all the precursors from this protein identification run, as obtained from the descriptive stats template that is provided with ProteinPilot software

Here, the delta ppm error for this dataset was 1.48 RMS

This high resolution is maintained in the MS/MS spectrum as well

Shown here is a peptide from one of the E. coli proteins. And you can see that resolution in MS/MS mode is very nice, typically greater than 25,000 resolution across the mass range, and even at the very low end of the mass range, the resolution is still high

So, we ran the same E. coli experiment on both the TripleTOF 4600 and 5600 systems to assess the performance differences between the two platforms

All data was processed using ProteinPilot software and compared at the same false discovery rates, using 5 percent local FDR at the peptide level and 1 percent global FDR at the protein level

The first comparison we did was to compare both platforms using unit resolution isolation on the Q1 mass analyzing quadrupole

The green text and points shows the data from the 5600 system, and the red dots shows the 4600 data

You can see there's about a 20 percent reduction in peptide detections on the TripleTOF 4600 system versus the 5600 system, which amounted to about roughly 10 percent fewer proteins

Because the sensitivity is expected to a bit lower on the 4600, we then repeated the experiment using a more open Q1 setting, low resolution on Q1

This provided a small increase in detected peptides, as expected, about 10 percent more peptides over unit resolution and is shown by the blue text and dots

These results demonstrate that the TripleTOF 4600 system is a great platform for protein ID due to its high sensitivity and acquisition speed

Because of the high resolution and high acquisition rates, the TripleTOF 4600 system is also a solid system for doing discovery proteomics using MS/MS-based quantitation strategies, such as the iTRAQ reagent workflow

The resolution observed on the reporter ions is very high, typically greater than 20,000 resolution, which ensures that quantitation data is clear of any low-mass fragmentation interferences

I don't have time to go into all the details here, but we did a small benchmark study using iTRAQ reagents on the TripleTOF 4600 system and were very excited about the data we obtained

Known differential proteins were spiked into a complex background, and we analyzed the ability of the MS system plus software to automatically distinguish the real differential changes from analytical variations

We did this using a target decoy strategy. The top panel shows the ratios between the two analytical replicates for the same physical sample. This is our decoy, as we expect to detect no differences, and you can see that we see very few false detections

The bottom pane shows the ratios for the proteins in the different samples

Here, we see a number of statistically significant changes. And most of these correspond to the added differential proteins we expected

All of this data computation was done in the descriptive statistics templates provided with ProteinPilot software

The ProteinPilot Software was used for all this data processing and is a really excellent partner for the TripleTOF family of MS systems

Because of the speed of the TripleTOF 4600 system, we are digging much deeper into our proteomes

We therefore need a search engine that is able search for the many different low-abundant modifications and unexpected cleavages that we see when we do dig deeper into these complex samples

The Paragon algorithm is a very elegant solution for searching extremely large search space without exploding the number of false positives detected

Now, assembling all that peptide data into proteins is a crucial step in proteomics and biomarker discovery. And the ProGroup algorithm does a very rigorous analysis, ensuring that the numbers of proteins reported is not falsely inflated

Protein expression analysis is done automatically for many of the label-based quantitation strategies, both MS and MS/MS quantitation

And finally, false discovery rate analysis is built in, such that results can be communicated in a non-search-engine-specific format and be consistent with journal requirements

The next workflow I'd like to discuss is the high-resolution targeted peptide quant workflow called MRMhr workflow

In this analysis, the instrument is set up to acquire full-scan MS/MS data on a fixed precursor of interest over and over again in an LC run

The Q1 is fixed. The peptide is fragmented in the collision cell. And full-scan TOF MS/MS is acquired. This is repeated across the LC run

After data acquisition is complete, extracted ion chromatograms on sequence-specific ions can be generated

Multiple fragment ions can be monitored or even summed together because the full-scan MS/MS is always acquired

Here, the same rules apply as MRM. You want the cycle time such that six to eight points across the peak at half height are obtained for accurate peak integration

After this extraction, the processing for quantitation is now very similar to how one would handle MRM data, as acquired on a triple-quad or QTRAP instrument

A set of low-abundant E. coli proteins that were detected in the protein identification experiment explained earlier were targeted in a multiplexed MRMhr workflow

Example data for one of the peptides is shown here, with the experimental MS/MS shown on top and XICs for three fragment ions shown on the bottom

Because of the high sensitivity and speed of the TripleTOF 4600 system, accumulation times as low as 10 milliseconds can be used, which allows good multiplexing

However, we can obtain even higher multiplexing by scheduling the MRMhr workflow in time

This new workflow is available in the Analyst TF 1.6 software available for all TripleTOF platforms

When the retention time of the peptide is known, the acquisition method can be easily built by providing a list of peptides with their precursor M-over-Z and retention time, as shown on the left

The peptides are not monitored all the time. So, the accumulation times used for the acquisition of the full-scan MS/MS data can be longer for each peptide at its elution time. And this is shown by the plot on the right

This shows a scheduled MRMhr workflow on 1,000 targets, with a maximum concurrency of 50 targets per cycle

Finally, because the full-scan data is always acquired, the user can always adjust the fragment ions used for quantitation and the resolution used to extract the data post-acquisition

Because the TOF MS/MS data is very high resolution, narrower XIC windows can be used for extraction, which can reduce the general background noise and also specific interferences

This ensures the highest-quality data is obtained across the whole dataset

An example of background reduction is shown here, as the extraction windows are tightened from 0.7 width down to 0.02 Daltons

As the TripleTOF 4600 system a TOF-based system, it is an excellent system for the determination of protein molecular weights by analyzing intact proteins

Here, we typically run a short LCMS run on the protein to help clean off any associated salts. The charge state envelope is analyzed and is shown in the middle

The TOF MS has a mass range up to 40 kilodaltons, which will easily cover large proteins

With good sample clean up, a nice resolution can be obtained on the individual charge states, as shown in the top right, to determine if there are any modified forms of the protein

Finally, mass reconstruction using BioAnalyst software can be performed to determine the mass of the main protein form

This is an example of a large IgG protein

In conclusion, the new AB SCIEX TripleTOF 4600 system is a powerful new system for all of these proteomics and protein workflows

The ability to perform high-resolution and high-mass-accuracy MS scans with very high sensitivity enables detection and quantification of more peptides and proteins

From this TOF MS data, the TripleTOF 4600 system will acquire high-resolution MS/MS spectrum at very high-acquisition rates, driven by the very high sensitivity of the instrument in this MS/MS mode

Combine these powerful instrument attributes with the innovative data processing software and high-performance LC system from Eksigent, and you have a powerful accurate mass workhorse solution for protein identification and quantitation

Finally, I'd like to thank my colleagues at AB SCIEX for work on this project