Asn₃, a Reliable, Robust, and Universal Lock Mass for Improved Accuracy in LC–MS and LC–MS/MS

Abstract

The use of internal calibrants (the so-called lock mass approach) provides much greater accuracy in mass spectrometry based proteomics. However, the polydimethylcyclosiloxane (PCM) peaks commonly used for this purpose are quite unreliable, leading to missing calibrant peaks in spectra and correspondingly lower mass measurement accuracy. Therefore, we here introduce a universally applicable and robust internal calibrant, the tripeptide Asn3. We show that Asn3 is a substantial improvement over PCM both in terms of consistent detection and resulting mass measurement accuracy. Asn3 is also very easy to adopt in the lab, as it requires only minor adjustments to the analytical setup.

Elucidating Escherichia coli Proteoform Families Using Intact-Mass Proteomics and a Global PTM Discovery Database

“…Intact-mass data were collected by LC-MS analysis on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer without fragmentation. Each fraction was injected twice (two technical replicates), using an AGC target setting of 10 6 , with a maximum fill time of 500 ms. A lock-mass standard (penta-asparagine) was added to the chromatographic buffer for continuous mass calibration 13 (Supplementary Experimental Methods). Three biological replicates were performed, yielding a total of 58 files of mass spectra (in .raw format).…”

Ring Finger Protein 213 Assembles into a Sensor for ISGylated Proteins with Antimicrobial Activity

“…MS/MS spectrum data type was set to centroid. The polydimethylcyclosiloxane background ion at 445.12002 was used for internal calibration (lock mass) in addition to the ion at 361.14660 corresponding to a tri-peptide of asparagine that was spikein the MS solvents as described in (Staes et al, 2013). Similar settings were used for the GST pull down and immunoprecipitation experiments.…”

Elucidating Proteoform Families from Proteoform Intact-Mass and Lysine-Count Measurements

“…The false discovery rate is expected to drop with improvements to instrument stability, such as the utilization of a lock-mass standard in the chromatographic buffer for continuous mass calibration. 27 …”