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Rapid Liquid Chromatography and Fast MS/MS Acquisition Enables High Throughput Protein Identification Workflows
LC/MS/MS is now a standard method for the analysis of protein samples that have been separated by 1D or 2D gel electrophoresis with subsequent in-gel digestion. However, the throughput tends to be low for this workflow since the analysis typically requires the use of longer gradients and data acquisition times of over 30 minutes per sample. Here we show very rapid LC gradients - as fast as 1 minute - using monolithic columns combined with intelligent, efficient data acquisition to provide protein identification results that are equivalent to standard gradient separations.
Overview of the QSTARŽ Elite SystemImprovements in hardware, electronics, and software to the QSTARŽ Elite System vs. the previous model QSTARŽ XL System enables much faster acquisition of MS/MS data. New software features, such as Smart Exit and Smart CE, take advantage of this speed improvement and provide a new level of intelligence in data acquisition. With a minimum accumulation time of 100 msec, decisions about MS/MS signal/noise and fragmentation quality can be made on-the-fly to provide higher quality data as well as more data overall.
The TempoTM nano LC system employ a direct pumping system with independent flow rate monitoring for each mobile phase, and is capable of delivering a precision of <1 nL/min before mixing. This enables precise, rapid gradient formation. Close coupling of the pump, gradient mixer and autosampler minimizes dispersion and delay time enabling very fast LC runs. New ProteinPilotTM Software combines two state-of-the-art algorithms to perform thorough, reliable, and easy-to-use protein identification; Mascot (Matrix Sciences) and the ParagonTM Algorithm (Applied Biosystems). The novel ParagonTM Algorithm for database searching can efficiently consider about 150 modifications and unexpected cleavages to identify peptides from MS/MS spectra. The Pro GroupTM Algorithm performs a statistical analysis on the peptides found to determine the minimal set of confident protein identifications.
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Key Advantages for Rapid Protein IdentificationMethods
Sample: Bovine serum albumin (Michrom BioResources) was digested with trypsin and injected at a level of 100 fmol of digest on column.
Chromatography: Very rapid nanoflow chromatography was performed using the TempoTM nanoLC system. A Chromolith column (Merck, 100 ėm ID) was run using a 1 minute gradient at a flow rate of 800 nL/min. The total LC method time including re-equilibration from injection to injection was 6 minutes (Figure 2).
Mass Spectrometry: LC/MS/MS analysis was performed using the NanoSprayŽ II source and Heated Interface on the QSTARŽ Elite LC/MS/MS System. Using the new Smart IDA (Information Dependent Acquisition) features within AnalystŽ QS 2.0 Software, extremely fast and intelligent MS/MS acquisitions were performed.
Database searching: Protein identification was performed using the ParagonTM Algorithm in ProteinPilotTM Software or Mascot. Sequence coverage was automatically calculated within the software.
The QSTARŽ Elite System Provides Much More Protein Sequence CoverageTo illustrate the increase in data quality and quantity, 100 fmol of BSA tryptic digest was injected on a 100 ėm ID column running at an extremely fast gradient of 1 minute. Data were acquired on both a QSTARŽ XL and QSTARŽ Elite System. On the QSTARŽ Elite System, a Smart Exit setting of 1.0 was used, resulting in a typical MS/MS scan time range of 0.2-1.5s. As the QSTAR XL system originally provided either Mascot or Pro ID Software, Mascot was used to process the data. The QSTARŽ Elite System is provided with both the Mascot and Paragon Algorithm. Overall, for the QSTARŽ Elite System, the number of unique peptides confidently identified using the Paragon Algorithm increased >4x (Figure 1) vs. the QSTARŽ XL System using Mascot. This corresponds to a sequence coverage increase from 20% to 50% on the QSTARŽ Elite System.
This improvement in data coverage was reproducible, as observed by similar increases in sequence coverage for 10 replicate injections for the QSTARŽ Elite vs. QSTARŽ XL System (Figure 3, left), in this case using ProteinPilotTM Software on both datasets for ease of comparison. An effective doubling in the Unused ProtScore (ProteinPilotTM Software protein confidence measure) was obtained on the same dataset (Figure 3, right).The Unused ProtScore is a measure of the increase in evidence specific to each identified protein. This measure takes into account the confidence of each unique peptide identification, with higher confidence peptides contributing more to the protein's Unused ProtScore.
Automatic Instrument Calibration (AutoCal) Provides Stable High Mass Accuracy
Another key element that factors into the confidence of peptide identification is the mass accuracy of the peptide molecular weight. A new software feature, enabled by the improvements within the QSTARŽ Elite system is AutoCal, the automatic instrument calibration feature. This feature can perform an internal calibration of the instrument on a spectrum-by-spectrum basis during LC/MS acquisition. Coupled with the file recalibration features within ProteinPilotTM Software, much smaller mass tolerances can be employed during a database search, thereby increasing the confidence in identification and decreasing the false positive rate. Shown in Figure 4 is the average mass error on the identified peptides in each of the LC/MS runs. The majority of the LC runs have average mass errors below 6 ppm.
Ultra Fast LC Gradients Can Provide Equivalent Sequence Coverage
To investigate even faster modes of MS/MS acquisition, the Smart Exit Factor was lowered to a setting of 0.1. In this case, the average time of MS/MS acquisition was varied between 0.14 and 0.4s, depending on the intensity of the precursor ion.
Utilizing the most rapid Smart Exit setting on the QSTARŽ Elite system, the sequence coverage obtained on the 100 fmol injection of BSA tryptic digest increased from 60% to 70% (Figure 5). The ProtScore of the LCMSMS run also increased from 40 to 61using the faster acquisition rate.
The QSTARŽ Elite system is able to perform the high speed MS/MS scans without the loss in quality by implementing real-time monitoring of the MS/MS spectra. Termed Smart Exit, each MS/MS acquisition is stopped when the fragmentation spectrum reaches a user-definable quality level. This results in a variable accumulation time for each MS/MS spectrum. Thus, acquisition exits more rapidly on the more intense precursors, and acquires longer for the weaker ions.
High Sequence Coverage Obtained on Many Proteins
To further illustrate this improvement in speed and data quality, a number of other simple protein digests were analyzed (Table 1). A high sequence coverage and Unused ProtScore was obtained on all proteins analyzed using this extremely rapid 1 minute LC/MS acquisition.
MS/MS Data Quality is Maintained at High Acquisition Speeds
It is essential to maintain high data quality as the speed of MS/MS acquisition increases. Shown in Figure 6 is an MS/MS spectrum for an alpha casein peptide acquired at an exit factor of 1.0, a total MS/MS acquisition time of 180 ms. The high quality fragmentation pattern allows for the unambiguous assignment of the peptide as well as the site of phosphorylation. During the Hemoglobin acquisition, peptide MS/MS was obtained during a 2.2 minute time window. 72 MS/MS spectra were acquired, 59 of which produced a peptide identification yielding efficiency was extremely high - >80% - and most MS/MS acquired during the elution time of the peptides yielded a peptide identification. This highlights the in data acquisition used by the IDA acquisition software. There was still a significant portion of time when no MS/MS was acquired indicating that the IDA acquisition was operating at maximum speed.
Conclusions
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Š 2006 Applera Corporation and MDS Inc. All rights reserved. Applied Biosystems is a registered trademark and AB (Design) and Applera are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. Tempo, ProteinPilot, Pro Group and Paragon are trademarks and Analyst, QSTAR and NanoSpray are registered trademarks of Applied Biosystems/MDS SCIEX, a joint venture between Applera Corporation and MDS Inc. MDS and SCIEX are registered trademarks of MDS Inc.
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