A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples.

Abstract:

:Affinity purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear, however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is useful for reducing sample complexity prior to MS analysis and increases peptide and protein coverage of complex samples. Here, we monitored the effects of upstream protein or peptide separation techniques on typical mammalian AP-MS samples, generated by FLAG affinity purification of four baits with different biological functions and/or subcellular distribution. As a first separation step, we employed SDS-PAGE, strong cation exchange LC, or reversed-phase LC at basic pH. We also analyzed the benefits of using an instrument with a faster scan rate, the new TripleTOF 5600 mass spectrometer. While all multidimensional approaches yielded a clear increase in spectral counts, the increase in unique peptides and additional protein identification was modest and came at the cost of increased instrument and handling time. The use of a high duty-cycle instrument achieved similar benefits without these drawbacks. An increase in spectral counts is beneficial when data analysis methods relying on spectral counts, including Significance Analysis of INTeractome (SAINT), are used.

journal_name

Proteomics

journal_title

Proteomics

authors

Dunham WH,Larsen B,Tate S,Badillo BG,Goudreault M,Tehami Y,Kislinger T,Gingras AC

doi

10.1002/pmic.201000571

subject

Has Abstract

pub_date

2011-07-01 00:00:00

pages

2603-12

issue

13

eissn

1615-9853

issn

1615-9861

journal_volume

11

pub_type

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