Comparing monolithic and microparticular capillary columns for the separation and analysis of peptide mixtures by liquid chromatography‐mass spectrometry

Abstract

A mixture of ten proteins was trypsinized and injected onto poly-(styrene-divinylben-zene) monolithic columns (60 x 0.20 or 0.10 mm ID) and a column packed with C18 silica particles (75 x 0.075 mm ID), respectively. The columns were eluted at 200-2000 nL/min with gradients of ACN in 0.050% TFA. Eluting peptides were detected by ESI-MS/MS and subsequently identified by database searching. The 100 microm ID monolithic column showed the highest cumulative Mowse scores based on the highest ion scores for the peptides and the largest number of identified peptides. It is shown that the number of identified peptides strongly depends on the dynamic range within the peptide mixture. In consequence, all proteins were identified in a mixture of relatively balanced analyte amounts (12.5-80 fmol) whereas only peptides for six out of ten proteins were found in a sample of high-dynamic range (0.65-270 fmol). The 100 microm monolithic column showed the highest reproducibility for peptide identifications in three consecutive runs. Depending on sample amount, 57-72% of the identified peptides were detectable in each of the three runs of triplicate analyses. The results demonstrate the high suitability of 100 microm monolithic columns for high-resolution peptide separations in proteomic research.

Quantification of Hepcidin-25 in Human Serum by Isotope Dilution Micro-HPLC–Tandem Mass Spectrometry

“…(1 ). An earlier study found very good agreement between serum HCO 3 Ϫ values obtained with a Beckman Astra analyzer and those calculated from measurements taken with a Radiometer blood gas instrument (2 ). Thus, the degree of agreement between measured and calculated HCO 3 Ϫ values is likely a function of the particular methods used within the laboratory.…”

“…A stable isotope-labeled internal standard helps to compensate for any matrix effects. Micro-HPLC-MS/MS with monolithic capillary columns ensures highest resolution and limits of quantification (3 ).…”

“…Thus, the degree of agreement between measured and calculated HCO 3 Ϫ values is likely a function of the particular methods used within the laboratory. Some authors have also suggested that the pK value for HCO 3 Ϫ may vary substantially with the patient population (3,4 ).…”

De novo peptide sequencing by tandem MS using complementary CID and electron transfer dissociation

“…Mixture 1 comprised thyroglobulin (bovine thyroid gland) and albumin (bovine serum), whereas mixture 2 contained b-casein (bovine milk), conalbumin (chicken egg white), myelin basic protein (bovine), hemoglobin (human), leptin (human), creatine phosphokinase (rabbit muscle), a1-acidglycoprotein (human plasma), and albumin (bovine serum). The resulting peptide mixtures were then separated using capillary ion-pair RP HPLC (IP-RP-HPLC) and subsequently identified by ESI-MS/MS as described in detail in [32,33]. Briefly, one-microliter samples was desalted and preconcentrated at a flow rate of 10 mL/min of 0.05% aqueous TFA in a capillary/nano HPLC system (Model Ultimate, Dionex Benelux, Amsterdam, The Netherlands) using a 10.0 Â 0.2 mm monolithic poly-(styrene/ divinylbenzene) preconcentration column, which was prepared according to the published protocols [32,34].…”

Less common applications of monoliths

“…Many members of both Porapak and Chromosorb series GC packings are based just on this type of chemistry [42]. Monolithic versions of these polymers were also thoroughly studied in liquid chromatography [49][50][51][52][53][54][55][56][57]. The typical poly(divinylbenzene) monoliths used in HPLC separations of large molecules featured large through pores and no appreciable volume of small pores.…”