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J Biomol Tech. 2010 September; 21(3 Suppl): S52–S53.
PMCID: PMC2918147

Making a Case for Data-independent Tandem Mass Spectrometry Workflows

S. Chen, A. Panchaud, and D. Goodlett
Department of Medicinal Chemistry, University of Washington, Seattle, WA, United States



In tandem mass spectrometry data-dependent analysis (DDA), precursor ion detection is prerequisite to MS/MS, a condition that results in sampling a limited dynamic range and poor run to run identification reproducibility. We have improved this process by developing a data-independent analysis strategy coined PAcIFIC (Precursor Acquisition Independent From Ion Count). In PAcIFIC discrete regions of the mass spectrum (2.5 Da isolation windows) are interrogated sequentially by MS/MS. The limitation comes in matching the instrument duty cycle to fall within typical chromatographic peak widths, requiring several days of analysis time to cover a complete m/z range. Nonetheless, the method has shown dramatic improvement in the number of peptide identifications over data-dependent acquisition methods. An alternative is to perform PAcIFIC by direct infusion, allowing data acquisition in minutes rather than days. We applied this strategy to analysis of a bovine serum albumin (BSA) tryptic digest, both with and without prior C18 desalting, by direct infusion to an LTQ-FT (Thermo) mass spectrometer modified with an ion funnel. Mass spectra from un-desalted digests were devoid of multiply charged ions but gave similar sequence coverage to desalted samples at a range of concentrations. For example, at 1.0 pmol/μl, BSA sequence coverage was 68.5 % (± 1.1%, triplicates) for the desalted sample and 65.2 % (± 1.2%) for the un-desalted sample. Spectral counting yielded 97.9% and 91.4% overlap between runs for the desalted and un-desalted sample, respectively. Overall, the un-desalted sample gave 76.3% of the number of peptides corresponding to 93.9% of the spectral counts common to the desalted sample. By comparison, typical DDA LC-MS/MS gave similar identification results but with less reproducibility and longer analysis time (90 versus 8 minutes). For simple mixtures, we conclude that direct infusion using data-independent methods are fast and capable of performing well even in the presence of salt matrix.

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