With the fast growth of protein databases, there is also an increased demand for highly sophisticated mass spectrometry (MS) based methods for protein identification (protein ID).

Several aspects of experimental set-up can help to reduce or eliminate false positive and false negative results from protein mixture analyses.

1) Mass Accuracy: The mass of a peptide after digestion should be measured with the highest possible mass accuracy. The higher the accuracy of the result, the faster the database search will be and the lower the probability of a false positive or negative protein ID.  MS/MS fragmentation information is still required to confirm the database search result and identify the location of any post-translational modifications (PTMs).

2) Sequence Coverage: Proteins should be identified with highest possible sequence coverage.

3) Robust Separation Methods: To counter ionization suppression effects in the electrospray process, complex mixtures of protein digests should be purified and separated by HPLC prior to MS analysis.

4) Protein Coverage: All proteins in a mixture should be identified. Often the proteins of interest are those that are present at very low abundance.

The Finnigan LTQ FT is a hybrid mass spectrometer consisting of a linear ion trap and a FTICR instrument as a second mass analyzer. The two mass analyzers can measure a complex mixture in a concerted fashion using the advantages of each mass analyzer: the rapid and highly sensitive MS/MS capabilities of the linear ion trap and the extremely accurate mass measurement of the FTICR cell.

These features have been successfully combined in a parallel manner such that one MS spectrum is acquired with high resolution and high mass accuracy (< 2 ppm) in the FTICR cell at the same time that MS/MS experiments are taking place in the linear ion trap. The workflow for this parallel detection method is shown in Figure 1. In this illustration, LT represents the linear ion trap, FT represents the FTICR cell, and Processor represents the front end processor of the instrument.

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Further enhancement in detection can be accomplished by extension of the parallel detection method by utilizing a selected ion monitoring (SIM) method. The SIM method permits sub-ppm mass accuracy. The general workflow of the new SIM methods, illustrated in Figure 2, was first published by M. Mann et al.1
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Using the SIM method, the first MS spectrum acquired in the FTICR cell uses a very high target value to store a very large number of ions—more ions than in the standard parallel detection method.

Overfilling the ICR cell can lead to a decrease in mass accuracy, but it greatly enhances the dynamic range. In this way, very low abundant species can be detected and selected for Data Dependent™ fragmentation.

The FTICR spectrum obtained with a high target value is complemented by a Data Dependent precursor (SIM ) scan and a Data Dependent MS/MS scan.  The SIM scan is highly sensitive and allows very accurate mass measurement of the precursor ions on the order of 0.5 ppm or better.

With the SIM parallel detection approach, protein ID is based on ultra high accurate mass measurement of the precursor ions and their MS/MS fragments in the linear ion trap. A huge advantage of the ultra high accurate mass determination is a decreased average protein database search time. More importantly, low abundant species are detected by maximizing the storage capabilities of the instrument.  Finally, the full duty-cycle of this method permits on-line coupling with HPLC, with one set of parallel detection experiments generated per second.

To demonstrate the superiority of this approach, a 1D-SDS -PAGE band containing different proteins was digested in-gel, extracted and analyzed. With the standard parallel detection method, eight proteins could be unambiguously identified. The new SIM methods unambiguously identified ten additional proteins, resulting in the identification of 18 proteins in a single 1D-SDS -PAGE band.

1) Jesper V. Olsen, ShaoEn Ong and Matthias Mann; Mol. Cell Proteomics, 3, 608, 2004

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