Abstract
In this work, two online comprehensive two-dimensional liquid chromatography platforms, namely Hydrophilic interaction liquid chromatography × Reversed phase (HILIC × RP) and Reversed phase × Reversed Phase (RP × RP) coupled to mass spectrometry, were compared for the analysis of complex peptide samples. In the first dimension, a HILIC Amide and C18 Bioshell peptide (150 × 2.1 mm, 1.7 and 2.0 μm) columns were selected, while, in the second dimension, a short C18 (50 × 3.0 mm, 2.7 μm) Bioshell peptide column was used. Two C18 trapping columns (10 × 3.0 mm, 1.9 μm), characterized by high retention and surface area, were employed as modulation interface in both HILIC × RP and RP × RP methods. The LC × LC platforms were coupled to UV and tandem mass spectrometry detection and tested for the separation and identification of two gastro-intestinal digests of commercial microalgae formulations (Spirulina Platensis and Klamath). Their performances were evaluated in terms of peak capacity, maximum number and properties of identified phycocyanin peptides. Our results showed that the HILIC × RP approach provided the highest peak capacity values (nc HILIC × RP: 932 vs. nc RP × RP: 701) with an analysis time of 60 min, while the RP × RP approach was able to identify a slight higher number of phycocyanin derived peptides (HILIC × RP: 88 vs. RP × RP: 103). These results point out the flexibility and potential of HILIC × RP and RP × RP based on trapping modulation for peptide mapping approaches.
Highlights
Liquid chromatography coupled to mass spectrometry (LC-MS) represents the gold standard for the analysis of complex samples, such as in omics studies
We have recently demonstrated that the employment in the modulation interface of trapping columns, packed with highly retentive and efficient C18 sub-2 μm particles, with large surface area, delivered a sensible sensitivity increase with respect to conventional C18 trapping columns, showing a high potential in LC × LC-high resolution mass spectrometry (HRMS) for peptidomics [11]
When multiple enzymes are used, such as during the gastro-intestinal digestion, this situation is further complicated by subsequent cleavages, and the exact number of derived peptides cannot be calculated. 1D-LC-MS is unable to provide adequate peak capacity values and, as result the MS is overwhelmed by coeluting peptides entering into the MS at the same time
Summary
Liquid chromatography coupled to mass spectrometry (LC-MS) represents the gold standard for the analysis of complex samples, such as in omics studies. The transfer of the highly organic HILIC mobile phase onto the second RP dimension leads to heavy negative effects on peak shape (peak distortion or sample breakthrough) and a dramatic loss of sensitivity To circumvent these drawbacks, several strategies have been adopted in HILIC × RP, in particular, the employment of active solvent modulation, with and without trapping column-based modulators, in the analysis of partially digested monoclonal antibodies and histones [8,9]. “total breakthrough” conditions were used in HILIC × RP approach and compared to RP × RP approach, with a loop-based modulator, in terms of peak capacity [10] In this regard, we have recently demonstrated that the employment in the modulation interface of trapping columns, packed with highly retentive and efficient C18 sub-2 μm particles, with large surface area, delivered a sensible sensitivity increase with respect to conventional C18 trapping columns, showing a high potential in LC × LC-high resolution mass spectrometry (HRMS) for peptidomics [11]. For the reason stated above, cutting edge analytical methods able to elucidate in detail their peptidomic profile are mandatory
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