Two-dimensional Size Exclusion Chromatography Research Articles

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones.

Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Because of the extreme complexity of histones regarding the number of proteoforms, new analytical tools with high-capacity separation and highly sensitive detection of proteoforms are required for TDP of histones. Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified a comparable number of proteoforms to RPLC-MS/MS from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. Three μg), indicating its substantially higher sensitivity. We identified about 400 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We identified histone proteoforms carrying various tentative post-translational modifications (PTMs), for example, acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. The electrophoretic mobility (μef) of unmodified histone proteoforms can be predicted accurately (R2 = 0.98) with an optimized semiempirical model based on our recent work. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

The size dependence of the average number of branches in amylose

Amylose has a small but significant number of long-chain branches. Two-dimensional size-exclusion chromatography is used to obtain the first measurement of the average number of branches per amylose molecule (from potato tubers) as a function of molecular size. Molecular weight dispersity, average chain length and average amylose molecular weight all increase with increasing size. However, the average number of branches of amylose molecules is weakly, if at all, dependent on size, with 2–4 per molecule except perhaps for the very largest molecules, although for these, the data may suffer from artifacts. Differences in the sizes of amylose molecules is mostly ascribed to variations in chain length. This observation is consistent with the postulate that most branching events occur in the early stage of amylose synthesis, and afterwards the branches are further elongated by granule-bound starch synthases. This gives improved mechanistic understanding of amylose biosynthesis.

Evaluation of tangential flow ultrafiltration procedures to assess trace metals bound to marine dissolved organic matter

A procedure based on two-dimensional size exclusion chromatography (SEC) and anion exchange chromatography (AEC) with UV (205nm) and ICP-MS detection was used to assess dissolved organic matter (DOM) and trace metals associated to DOM in surface seawater. Marine DOM was isolated by tangential ultrafiltration (UF) using two different polyethersulfone membranes exhibiting different molecular weight cut-off (MWCO), 3 and 10kDa. The procedures require a volume of seawater sample of 100L, and marine DOM of molecular weight higher than 3 and 10kDa (UF membranes of 3 and 10kDa MWCO) was pre-concentrated in 0.5L (retentate), which implies a pre-concentration factor of 200. Retentate fractions obtained after the different UF procedures were further desalted by using HI Trap desalting mini-columns before two-dimensional SEC/AEC. Apparent molecular weights of isolated compounds after SEC with UV detection ranged from 1.5kDa (close to the permeable volume of the SEC column fixed by injecting vitamin B12) to 16 and 22kDa (UF membranes of 3 and 10kDa MWCO, respectively). Further AEC/UV characterization of SEC fractions showed a large group of macromolecules eluted at 4.5min, and small signals at shorter retention times (2.5 and 3.5min). In addition, AEC experiments of the isolated SEC fractions when using 10kDa MWCO UF membranes showed a group of substances eluted at high retention times (13min). SEC hyphenation with ICP-MS proved the existence of several trace elements (Ni, Co, Cu, Zn, Mn, Mo and Sr) bound to the isolated marine DOM. Mass balance studies after analyzing the retentate and permeate fractions for trace elements indicate good recoveries (close to 100%) for elements such as Mo, Sr, Ba and Zn when performing the UF with both 3 and 10kDa MWCO membranes. However, recoveries from 36 to 81% were obtained for the remaining studied elements after either UF procedure. SEC-ICP-MS experiments showed percentages of metals bound to the isolated marine DOM ranging from 0.055 and 0.077% (Zn) to 4.1 10-4 and 1.4 10-4% (Sr). © 2013 Elsevier B.V.