Application Note # LCMS-81 Introducing New Proteomics Acquisiton Strategies with the compact Towards the Universal Proteomics Acquisition Method Introduction During the last decade, the complexity of samples addressed in bottom-up proteomics studies has increased along with the instrument s capabilities to generate more and more detailed information from these complex samples. Instrument speed, sensitivity and mass accuracy have reached new levels, enabling the generation of more data from increasingly complex samples and to better cope with the advances in U-HPLC separation. Whilst slower than other architectures - like ion traps - at the beginning of the century, Q-TOF and UHRQ-TOFs have now reached new levels in both speed and sensitivity. These improvements allow Q-TOF systems to generate identification and quantification results which are fully comparable with the ones obtained from the latest bench-top FT-based instruments, while keeping the advantage of preserved resolution at high acquisition speeds. Whatever technology is used, the number of unproductive spectra is relatively high, typically in the range of 60-70% of all spectra for a human cell line digest. The impossibility to link these spectra to a known protein most often comes from the occurrence of unexpected modifications, inaccurate database information or from spectra of poor quality. The two first reasons can be addressed by adjusting Authors Dr. Stephanie Kaspar, Dr. Markus Lubeck, Dr. Alexander Harder Bruker Daltonik GmbH, Bremen, Germany Pierre-Olivier Schmit Bruker Daltonique S.A, France Keywords compact impact Proteomics Biomarker Discovery Instrumentation and Software compact impact DataAnalysis ProteinScape ProfileAnalysis
the search method or performing de-novo sequencing, however trying to minimize the number of garbage spectra was always a matter of compromise between the acquisition speed and the number of MS points in the chromatogram (defining the Qual/Quant capabilities of a system). Moreover, if one compromise was valid for a sample with a given dynamic range and average concentration it wasn t the best option if these conditions were changed. With the compact Benchtop ESI-QTOF, we introduce a new release of the Compass acquisition software. It enables a series of new acquisition strategies that make it possible to use a single acquisition method to deliver optimal results, whatever the sample s initial concentration or dynamic range. The instrument s improved robust hardware enables this new benchtop system to deliver state-of the art results in bottom-up proteomics. This improved hardwaretogether with the new software features will also be available on the UHR-Q-TOF impact. Experimental The human HeLa cell line was digested using trypsin. 10-1000 ng lysate were injected into a nano LC system equipped with a Dionex Pepmap C18 25cm x 75µm column (2µm particles) coupled to a compact Benchtop Q-TOF or an impact benchtop UHR-QTOF both running with the new Compass 1.6 Acquisition and processing software. Both systems were operated with an automated internal re-calibration, and once acquired the data was processed with Bruker s ProteinScape bioinformatics software suite. Database searches were performed with Mascot 2.4 triggered by ProteinScape. The accepted peptide False Discovery Rate was set to 1%. Results and Discussion Compass 1.6: improved auto MS/MS acquisition strategies In the past, tuning the ideal auto MS/MS method has always been a matter of compromise: Compromise between identification results and quantification capabilities: only 3 to 4 MS per chromatographic peak are required to sample efficiently the precursor peaks population, but at least 6-7 are required to properly define the peak and make it possible to use it for accurate quantitation. The number of points were not predictable as directly defined by the number of precursors chosen between each MS/MS (20 for instance for a top 20 method) and by the time requested to perform each MS/MS. Compromise between the spectral quality and the acquisition speed: whatever active exclusion strategy used, the choice was either to favor the spectra quality or the total number of precursors from different species. The same was true for the acquisition s speed choice. A fast MS/MS acquisition speed favors the fragmentation of a higher number of species but degrades the spectral quality obtained from lower intensity precursors. The IDAS (Intensity Dependent Acquisition Speed) and RT 2 (RealTime Re-Think) functionalities of Compass 1.6 now make these compromises unnecessary. They consist of the following: with IDAS, the cycle time is fixed instead of the number of precursors selected for MS/MS, and the acquisition speed is adapted depending on the precursor intensity (Fig 1). This way, the number of precursors is directly adapted to the available precursor intensity, avoiding wasting time with unnecessary MS survey scans between many intense precursors and preventing missing low-intensity compounds if the acquisition is slowed down for very low abundant samples. Moreover, as the number of MS points defining a chromatographic peak is stable, the Qual/Quant capabilities of the instrument are maintained over the entire chromatographic run. Finally, the only parameter requiring adaptation is the time spacing of MS survey scans, which depends directly of the chromatographic performances delivered by the LC system used with the instrument. However, the IDAS does not handle the issue of inappropriate exclusion strategies, which can result in the same peptide being selected for fragmentation a number of times where its intensity has not improved between the different points, and consequently losing the opportunity to fragment a peptide belonging to another species. This situation is addressed by the RT 2 (RealTime Re-Think) functionality. RT 2 is a dynamic adaptation of the exclusion strategy (Fig 2): once a precursor is fragmented it is excluded for the typical time of a chromatographic peak unless its intensity increases by more than a defined factor (typically set to 5). This ensures, that a precursor is only re-fragmented if a substantial gain in spectral quality is to be expected from a second analysis. Other algorithms, which try to anticipate the peak s maximum and trigger an MS/MS selectively at that time, require well described peaks of reasonable intensity and tend to completely miss low abundant precursor ions appearing only in a low number of MS scans. RT 2 is the most robust approach here.
Intensity Dependant Acquisition Strategy 5.00 40 39 27 30 4.00 3.00 Auto cycle time [s] 2.00 1.00 0.00 MS MS MS MS 1 2 3 4 Auto cycles 20Hz 18 19 2Hz spectra rate Figure 1: IDAS: Illustration showing an example of time cycle handling with the Independent Data Acquisition Strategy: the number of precursors is chosen dynamically to maintain the cycle time constant and the MS/MS spectra rate is adapted to each precursor intensity. RT 2 : RealTime Re-Think A Figure 2: RT 2 : RealTime Re-Thinks management of the Dynamic exclusion. The basic exclusion time is fixed to slightly longer than the base width of the chromatographic peak. Case A: the MS/MS is triggered near the peak maximum the precursor is not fragmented again as intensity is not increasing by the factor set for RT2. Case B: the MS/MS is triggered a first time near the peak base theprecursor is reconsidered for fragmentation only if its intensity has increased (typically by 3-5 fold). B Global proteomics ID results obtained with the compact from a complex protein digest The new compact system powered by Compass 1.6 has been evaluated on a HeLa human cell line digest. A 20 ng injection of the sample separated and analyzed with IDAS and RT 2 activated for a 2-20Hz dynamic method resulted in the identification of 631 proteins at a 1% peptide FDR. The same method used with 3µg of the same sample and a 4h gradient resulted in 2575 ID s (Fig 3). This result is comparable to what could be achieved with a 2µg injection and a 3 hours gradient on an impact system operating with a comparable acquisition method but without IDAS and RT 2. The compact s typical resolution is 23000 @ 1222 m/z whereas the impact s is 40000 at the same mass. That means that the improvement in the parent selection combined with the improved spectral quality have partially compensated for the loss of resolution and almost driven the compact s ID potential to the formers impact level. Moreover, the gain in spectral quality obtained thanks to the IDAS and RT 2 combination makes it possible to drive the MS/MS spectrum ID rate from a 35% level to a 50% level. This translates into an on average higher quality of spectra obtainable, as illustrated in (Fig 4). Thus these new features further increase the already high spectral quality delivered by the instrument (Fig 5). The same level of gains could be obtained from the impact when used with the new acquisition strategy.
compact: ID s from complex mixtures m/z 1400 Instrument compact with new SW 1200 Sample (Hela) 3µg 20ng Gradient 4h 90min 1000 ID @ 1% FDR 2575 631 800 600 400 25 50 75 100 125 150 175 200 225 Time [min] Figure 3: compact ID s from complex mixtures. The survey view is issued from the separation and acquisition of 3µg of a HeLa cell lysate tryptic digest on a 50cm column with a 4h gradient. The resulting number of ID s was 2575@ 1% FDR. compact: spectral quality @ 20 Hz Figure 4: Focus on the Compact spectral quality @ 20Hz MS/MS acquisition rate. Mascot view of two deconvoluted ions acquired @ 20Hz from a 3 µg injection of a HeLa cell lysate separated on a 240min gradient. compact spectral quality: broad mass transfer Intens. 250 394.207 +MS2(973.648), 43.6-52.3eV, 92.2-94.7min #13956-14251 200 150 100 50 0 Pro 70.062120.079 181.095 280.165 442.274 493.277 628.337 743.368 707.341 658.353 592.314 913.468 842.437 1026.549 990.531 1141.588 1500 m/z 1435.707 1288.647 200 400 600 800 1000 1200 1400 m/z Figure 5: Display of the intrinsic spectral quality of the compact (further enhanced by IDAS and RT 2 ): this spectrum particularly illustrates the instrument s broad mass range transmission, which is ideal for qual/quant with isobaric tags (itraq/tmt).
Universal method : illustration with the impact The results obtained for various amounts of the HeLa human cell digest on an impact using a 90min gradient are displayed in (Fig 6). A common method was used for these samples, meaning that the dynamic range created by the distribution of the injected amounts adds to the sample s own dynamic range. The benefit of IDAS and RT 2 combination is that the system automatically adapts the acquisition rate to the amount of sample available, preserving both the spectral quality for optimal ID rates and the chromatographic resolution for qual/quant purposes. The preserved spectral quality is well illustrated by the relatively small drop in the average Mascot ions score between different sample amounts injected. The overall increase in performance is evident at all levels where the 2087 and 2097 ID s obtained for the two distinct 1µg injections represent roughly a 10% improvement in comparison with the same amount injected on the system without IDAS and RT 2. For this amount injected the percentage of significant hits increases from a 35% to a 54 % level. The 35% significant hits level is obtained with an injected amount of 100ng, that is for a 10-fold decrease injected amount. Conclusion The compact Q-TOF and impact UHR Q-TOF s state of the art capabilities for protein ID s, issued from their combination of robustness, speed, sensitivity, resolution and mass accuracy, are further improved by the use of the new IDAS and RT 2 protocols available in Compass 1.6. In addition to improving the method s usability for samples showing different complexities, this approach ensures a better global spectral quality, translated in higher Mascot ion scores and larger proportion of identified spectra. This versatility is enhanced as the method makes it possible to preserve optimal chromatographic profiles for every analysis, therefore enabling accurate quantitation. The new Compass 1.6 features make it possible to increase the global proteomics usability and capabilities of both compact and impact systems. The full scope of these capabilities will be exploited through the ProteinScape Bioinformatic suite that enable the extraction of useful biological information from the high quality data generated. These first results underline a very positive influence of the new precursor selection management. However, there is still room for improvement in managing the different charge states of a same compound present under a chromatographic peak. Use of universal method with impact Figure 6: Use of Universal method with the impact. Results issued from the analysis of 10 to 1000 ng of a HeLa cell lysate tryptic digest with a 90min gradient. The number of ID s @ 1% FDR are obtained with percolator, the average mascot ion scores are displayed without percolator.
Bruker Daltonics is continually improving its products and reserves the right to change specifications without notice. Bruker Daltonics 03-2013, LCMS-81, 1817862 Further information [1] Bruker App-Note LCMS-67 3,500 Proteins Identified from a Human Cell Lysate Using Complementary MALDI and ESI Data [2] Bruker Tech-Note TN-27 ProteomeQuant A Highly efficient Solution for Biomarker Discovery and label-free Identification of Regulated Proteins in Biological Systems [3] Bruker Tech-Note TN-43 Targeted Proteomics Using HR-XIC Filtering with Skyline For research use only. Not for use in diagnostic procedures. Bruker Daltonik GmbH Bremen Germany Phone +49 (0)421-2205-0 Fax +49 (0)421-2205-103 sales@bdal.de www.bruker.com Bruker Daltonics Inc. Billerica, MA USA Phone +1 (978) 663-3660 Fax +1 (978) 667-5993 ms-sales@bdal.com Fremont, CA USA Phone +1 (510) 683-4300 Fax +1 (510) 490-6586 ms-sales@bdal.com