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Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics *

View Article: PubMed Central - PubMed

ABSTRACT

Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed “spider fractionator” in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2–96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 μg into the sub-microgram range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid automated and in-depth characterization of 12 different human cell lines to a median depth of 11,472 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use, and facilitates comprehensive proteome characterization with minimal sample requirements.

No MeSH data available.


Related in: MedlinePlus

Comparison of pooled and non-pooled peptide mixtures and separation efficiency.A, total ion current of separately collected, 90-s elution cuts from the 1st dimension column. B, total ion current of automatically pooled fractions corresponding to the ones in A. C, histogram of peptides containing at least 75% of their total mass over all fractions in the indicated number of fractions.
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Figure 2: Comparison of pooled and non-pooled peptide mixtures and separation efficiency.A, total ion current of separately collected, 90-s elution cuts from the 1st dimension column. B, total ion current of automatically pooled fractions corresponding to the ones in A. C, histogram of peptides containing at least 75% of their total mass over all fractions in the indicated number of fractions.

Mentions: With the column connected to the Spider fractionator, we first collected each of 54 fractions (90 s duration) in their own tubes. Starting from fraction three, we chose every 8th fraction and analyzed these fractions separately in 100 min gradients on the 40 cm analytical column. The 90 s elution windows from the first dimension eluted roughly in the same region as expected if they had been separated on a low pH analytical column except that their elution range was expanded considerably due to the different pH values (Fig. 2A). However, generally the bulk of the peptides was still concentrated within only about 20–50% of the total gradient.


Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics *
Comparison of pooled and non-pooled peptide mixtures and separation efficiency.A, total ion current of separately collected, 90-s elution cuts from the 1st dimension column. B, total ion current of automatically pooled fractions corresponding to the ones in A. C, histogram of peptides containing at least 75% of their total mass over all fractions in the indicated number of fractions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383787&req=5

Figure 2: Comparison of pooled and non-pooled peptide mixtures and separation efficiency.A, total ion current of separately collected, 90-s elution cuts from the 1st dimension column. B, total ion current of automatically pooled fractions corresponding to the ones in A. C, histogram of peptides containing at least 75% of their total mass over all fractions in the indicated number of fractions.
Mentions: With the column connected to the Spider fractionator, we first collected each of 54 fractions (90 s duration) in their own tubes. Starting from fraction three, we chose every 8th fraction and analyzed these fractions separately in 100 min gradients on the 40 cm analytical column. The 90 s elution windows from the first dimension eluted roughly in the same region as expected if they had been separated on a low pH analytical column except that their elution range was expanded considerably due to the different pH values (Fig. 2A). However, generally the bulk of the peptides was still concentrated within only about 20–50% of the total gradient.

View Article: PubMed Central - PubMed

ABSTRACT

Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed “spider fractionator” in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2–96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 μg into the sub-microgram range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid automated and in-depth characterization of 12 different human cell lines to a median depth of 11,472 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use, and facilitates comprehensive proteome characterization with minimal sample requirements.

No MeSH data available.


Related in: MedlinePlus