Quality control based on isotopic distributions for high-throughput MALDI-TOF and MALDI-FTICR serum peptide profiling

Abstract

In this study, we have implemented a new quality control (QC) parameter for peptide profiling based on isotopic distributions. This QC parameter is an objective measure and facilitates automatic sorting of large numbers of peptide spectra. Peptides in human serum samples were enriched using reversed-phase C(18)-functionalized magnetic beads using a high-throughput robotic platform. High-resolution MALDI-TOF and ultrahigh resolution MALDI-FTICR mass spectra were obtained and a workflow was developed for automated analysis and evaluation of these profiles. To this end, the isotopic distributions of multiple peptides were quantified from both MALDI-TOF and MALDI-FTICR spectra. Odd peptide isotope distributions in TOF spectra could be rationalized from ultrahigh resolution FTICR spectra that showed overlap of different peptides. The comparison of isotope patterns with estimated polyaveragine distributions was used to calculate a QC value for each single mass spectrum. Sorting these QC values enabled the best MALDI spectrum to be selected from replicate spots. Moreover, using this approach spectra containing high intensities of polymers or other contaminants and lacking peptides of interest can be efficiently removed from a clinical dataset. In general, this method simplifies the exclusion of low quality spectra from further statistical analysis.

Immunochemical and Mass-Spectrometry–Based Serum Hepcidin Assays for Iron Metabolism Disorders

“…In our hands, sample throughput was higher for c-ELISA than for MS assays. In theory MS methods, however, can also be high throughput by automation (30,31 ). This technique requires robust protocols, because hepcidin readily sticks to laboratory plastics, especially with the use of small sample volumes.…”

Improved classification of breast cancer peptide and protein profiles by combining two serum workup procedures

“…Second, 5 μl of serum (from the same aliquot) was used for peptide enrichment with RPC18-functionalized MBs (Jimenez et al 2007; Nicolardi et al 2010). For this purpose, 10 μl of RPC18 Dynabeads (Invitrogen) was used for the analysis of 5 μl human serum.…”

“…In the case of RPC18 profiles, peak intensities were determined for each individual isotope of the 42 selected peptide clusters. This detailed information was used for quality control (QC) based on isotopic distribution as reported previously (Nicolardi et al 2010). For the purpose of this study, the peak intensities of individual isotopes were summed for each isotopic cluster, resulting in a single intensity value for each peptide.…”

Hepcidin in Human Iron Disorders: Diagnostic Implications

“…Furthermore, sample throughput is likely to be higher for ELISA than for MS assays. However, MS methods can also be automated (101,102 ), and would have to be optimized for hepcidin because its amphiphatic character makes it readily stick to laboratory plastics, especially when relatively small sample volumes are used in large tubes.…”