Cross-Hit Analysis: Reducing False Positives in AS-MS by Expanding the Well Search Window

Affinity selection mass spectrometry (AS-MS) is a powerful technique, but without careful data processing, false positives can overwhelm meaningful results.  One of the most overlooked factors is the scope of your cross-well background hit analysis.

Experiment

In this case study, using a 250,000-compound library across 96 wells, Virscidian's team demonstrated that limiting background hit analysis to the traditional “5 wells before and after” approach missed significant background interference.

Key Finding

As the search window was expanded across the entire 96-well plate, the number of confirmed hits dropped by 33%, indicating that nearly one-third of the original “hits” were in fact false positives due to recurring background noise.

Recommended Practice

Manual review of AS-MS data for cross-hits is inherently limited in scope and speed. Automated cross-hit analysis using Virscidian’s Analytical Studio dramatically improves the reliability of hit identification by detecting recurring background peaks and objectively applying consistent thresholds across the plate.  Using automated cross-well background hit analysis across the entire plate, not just a few surrounding wells, dramatically increases confidence in the final hit list and conserves resources by reducing false leads.