Quantitative proteomics often incorporates the use of stable isotope labels (SILs) to provide absolute quantification. Recent advancements have seen the introduction of peptide panels allowing the quantification of over 500 proteins in plasma sample sets. However, this is technically challenging when attempting to acquire the data using more traditional MS acquisition modes such as multiple reaction monitoring (MRM), since the duty cycle of the instrument is compromised and therefore results in under sampling. An alternative approach is to apply a data independent analysis (DIA) methodology, allowing for high throughput whilst ensuring high rates of data acquisition and specificity. We describe the use of a SIL kit capable of quantifying >500 plasma-based proteins in conjunction with SONAR, to quantify proteins of interest for patient cohorts diagnosed with respiratory disorders.
Undepleted human plasma originating from controls and patients diagnosed with chronic obstructive pulmonary disorder (COPD) and asthma were reduced, alkylated and tryptically digested overnight. Prior to LC-MS analysis, samples were spiked with PQ500 SIL peptides (Biognosys). Samples were separated using a 1mm scale column over various LC gradients (15, 30 and 60 minutes). MS data were acquired using SONAR, whereby the quadrupole (MS1) was continuously scanned between m/z 400-900 using a quadrupole transmission width of approximately 20 Da, whilst the TOF scanned between m/z 50-2000. In all cases, precursor and product ion information were collected. Data were processed with Spectronaut Pulsar X (Biognosys AG and Progenesis QI for Proteomics (Non-linear Dynamics). Acquiring data over a 1mm scale demonstrates greater robustness and highlights flexibility of the workflow for multi-OMICS based applications. Multivariate statistical analysis showed distinct differences between all three cohorts and proteins corresponding to >86% of the spectral library were quantified. CV’s for each group were all found to be <10% in all cases and was maintained at high throughput (i.e. 15 minutes).