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Two dimensional liquid chromatography (2D-LC) has long been a commonly used technique
in bottom-up proteomics, and is as relevant today as ever [1, 2]. To maximize the number of
protein identifications, analytical chemists have strived to obtain 2D-LC systems with a high
degree of orthogonality, which is needed to reach the required high peak capacities. A system
utilizing reversed-phase chromatography (RP) in both column dimensions with a large
difference in pH has proven to yield a higher peak capacity for peptides, even with a lower
degree of orthogonality compared to other combinations [3].
The present study is based on Reubsaet, et al. [4]. The study is a comprehensive offline RPRP 2D-LC system with high pH (>9) in the first dimension and low pH (3) in the second
dimension. The method’s purpose is to maximise protein identifications from one trypsinated
protein sample. To achieve maximum identifications, Reubsaet incorporated the concept of
concatenation of fractions. In practice, concatenation is applied through collecting the column
output from the first dimension in eight numbered vials, where the 1st, 9th, 17th, … fractions
are collected in the first vial, the 2nd, 10th, 18th, … are collected in the second, etc. [4].
In the present study, the fractionation scheme is examined by changing the total number of
vials in which samples are concatenated, the number of fractions in each vial, and the
collection time for each fraction. As the method demands high precision and is very timeconsuming, automatization is practically essential. Automatization is achieved with the spider
fractionation scheme, proposed and shown by Kulak, et al.[5]. Here, a 10-port injector
connects the first dimension column to each sample vial, and switches between them
automatically, reducing the strain on the operator drastically, while removing human errors.
The optimization of the concatenation will lead to higher numbers of identified proteins in
proteomics.
