Improvement of Recovery and Repeatability in Liquid Chromatography−Mass Spectrometry Analysis of Peptides

Abstract

Poor repeatability of peak areas is a problem frequently encountered in peptide analysis with nanoLiquid Chromatography coupled on-line with Mass Spectrometry (nanoLC-MS). As a result, quantitative analysis will be seriously hampered unless the observed variability can be corrected in some way. Currently, labeling techniques or addition of internal standards are often applied for this purpose. However, these procedures are elaborate and error-prone and may render complex samples even more complex. Moreover, whenever poor repeatability results from variable recovery, not just quantification, but also sensitivity is affected. We have studied the parameters influencing the repeatability of chromatographic peak areas for a model set of proteolytic peptides (i.e., a cytochrome c tryptic digest) in nanoLC-MS analysis. It is demonstrated that repeatability issues are mainly due to poor recovery of peptides from the sample vial. Problems are largely resolved by addition of an organic modifier to the sample vial to improve solubility of the peptides, but care needs to be taken not to lose peptides due to reduced affinity for reversed-phase materials. Good results are obtained when applying dimethylsulfoxide (DMSO) for this purpose. When applying DMSO, repeatability increases, and the limit of detection (LOD) decreases. For the most hydrophobic peptides, a gain in LOD of at least an order of magnitude is obtained. In an aqueous sample containing 0.1% formic acid (FA), it is possible to detect 100-200 fmol of peptide, whereas +/-10 fmol can be detected in a sample containing 5% FA and 25% DMSO (10 microL injections).

The Impact of Delayed Storage on the Measured Proteome and Metabolome of Human Cerebrospinal Fluid

“…Another study showed that blood contamination decreases the stability of the CSF proteome (11 ), corroborating our earlier results (21 ). One explanation for the decreased level of the 2 unidentified peaks in the proteome analysis is the possible adsorption to the vial surface, e.g., via hydrophobic or van der Waals interactions (21,33,34 ). Metabolomics revealed increased concentrations of threonic acid after storage at room temperature.…”

“…Concentrations of cystatin C and albumin were calculated on the basis of the measured ratios of the corresponding spiked isotope-labeled internal peptide standards to their biological counterparts, confirming that variation between the different time points was not statistically significant (see online Supplementary Tables S3 and S4). The measured concentrations of both proteins were both found to agree with reported CSF concentrations (32,33 ).The albumin concentrations measured by SRM were also in agreement with albumin concentrations measured by standard clinical chemistry techniques (Table 1). In addition, we assessed the trypsin cleavage efficiency by monitoring the release of a tag from the lysine end of the cystatin C peptide during the regular digestion procedure.…”

Presence of the Adenovirus IVa2 Protein at a Single Vertex of the Mature Virion

“…10% and are supplied as a dried film which may not be at the bottom of the vial. Peptide solubility is difficult to assess and is a major source of variation (36). In this study, the ability to calculate a molar extinction coefficient at 214 nm for specific peptides (22) allows correction for variation.…”

Longitudinal Assessment of Peptide Recoveries from a Sample Solution in an Autosampler Vial for Proteomics

“…According to previous reports, 12,17 "the average hydrophobicity of peptides is adequately expressed by RT in reversed-phase chromatography." Thus, the RT was used to separate peptides into the groups (RTs of hydrophobic peptides >35 min and RTs of hydrophilic peptides <35 min).…”

“…12 Calcitonin, a 32-amino acid hormone, is a well-known example of a peptide which adsorbs to glass and polypropylene. [13][14][15] Recently, Stejskal et al carried out three time-point comparisons of the number of peptides conditions; the combination of a polypropylene vial with a mixture of 0.001% polyethylene glycol (PEG) and 0.1% formic acid (or the mixture of 5 M urea, 1 M thiourea, and 0.1% formic acid) as a dissolution solvent showed the best performance.…”