Membrane-Assisted Isoelectric Focusing Device As a Micropreparative Fractionator for Two-Dimensional Shotgun Proteomics

Abstract

Recently, we introduced an online multijunction capillary isoelectric focusing (OMJ-CIEF) fractionator to fractionate proteins and peptides in electrospray-friendly solution. In this follow-up study, the original configuration of the fractionator was modified to improve the resolving power and reproducibility of separation. The major improvements include stabilization of the electrical current through the device using a voltage divider and stepwise elution of peptide zones in conjunction with the repeated refocusing of remaining peptides. Also, a novel algorithm was developed to calculate more accurately the pI values of peptides identified from experimental data. The standard deviation of calculated pI values for unmodified peptides from the theoretically predicted pI values was on average 0.21 pH units, which is more accurate than in standard-resolution gel-based methods. In order to characterize the analytical performance of the improved device, it was applied for the pI fractionation of yeast proteome digest into 18 fractions, with the collected fractions being analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry. Approximately 37% of 20047 identified peptides were detected in only one fraction and 27% - in two fractions. On average, every peptide was found in 2.4 fractions. These results strongly indicate the suitability of the improved device as a first dimension of separation in multidimensional shotgun proteomics analysis, with a potential for fully automated workflow.

Modified cysteine S-phosphopeptide standards for mass spectrometry-based proteomics

“…According to the classification proposed by Everley, all of the protonated pCys-peptides shown in Table 1 belong to a low proton mobility class of peptides, as their charge state z is equal to or lower than the sum of basic centers: (z ≤ Lys + Arg + His + 1) (Everley et al 2017). For this set of pCys-peptides, an interesting correlation was found between the calculated pI value (Pirmoradian et al 2014;Kozlowski 2016) of a plain peptide sequence and its NL-attribute. The pI criterion was used to test the set of low proton mobility peptides and revealed that NL-signals appeared in the HCD-spectra of the basic peptides incorporating the Arg-pCys unit but not in the spectra of acidic peptides which lacked this structural element ( Table 1).…”

Sample preparation in foodomic analyses

“…It can achieve much higher resolution of separation. Important advantage of CE and CIEF over gel‐based methods is that they can be directly hyphenated with a mass spectrometer .…”

“…However, MS‐based proteomic analysis still cannot access all proteome components since trypsin sequence specificity may not be suitable for detection . Increased proteome coverage was observed by combination of several proteases and cleavage strategies . The list of proteases used for digestion of proteins before LC‐MS/MS analyses is given in Table .…”

Isoelectric point region pI≈7.4 as a treasure island of abnormal proteoforms in blood

“…Recently we have introduced a multijunction capillary isoelectric focusing (MJ-CIEF) device that combines high sample capacity (up to 100 µg), ease and speed of operation (<1 h) with low sample losses (<10%) 17,18 . This device helped to increase the depth of the proteome analysis of tryptic peptide mixtures 17 and blood plasma proteins 18 . Here we employed the MJ-CIEF fractionator for testing the pI-pH hypothesis in serum of patients with neurodegenerative disorders.…”

“…One of the reasons for proteoforms to appear in that forbidden region is aggregation, a process characteristic for AD as well as other neurodegenerative disorders 22 . The novel MJ-CIEF device is applied in the current study together with the optimized proteomics techniques developed previously in our lab 17,18,23 . For testing the pI-pH hypothesis, patients from Western Norway diagnosed with probable AD and matched for gender (female) and age (77±6 years) were selected 24 .…”