Robust Microflow LC-MS/MS for Proteome Analysis: 38 000 Runs and Counting

Abstract

Microflow liquid chromatography tandem mass spectrometry (μLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 μL/min for high-throughput applications. On the basis of the analysis of ∼38 000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14 000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the μLC-MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and ∼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.

Figure 1. Summary of the number and types of samples analyzed on two micro-flow LC-MS/MS systems.

(A) Bar chart showing the number of samples analyzed by micro-flow LC-MS/MS on an Orbitrap HF-X (blue) and Orbitrap Lumos (red) mass spectrometer over time. The inset shows the total number of samples for each instrument. (B) Distribution of the types of samples analyzed by micro-flow LC-MS/MS on the Orbitrap Lumos. TMT: tandem mass tags; LFQ: label-free quantification; HpH: high pH reversed phase chromatography.

Figure 2

(A) Pie charts summarizing the number of samples analyzed using different columns on the Orbitrap HF-X and Lumos instruments. (B) Base peak chromatograms of 500 fmol of PROCAL peptides separated on the same PepMap column (1#) connected to the Orbitrap HF-X in the forward flow direction (upper panel; November 14^th 2019), reverse flow direction (middle panel; November 16^th 2019), and again in forward flow direction (bottom panel; August 3^rd 2020).

Figure 3. Chromatographic stability of the same PepMap column over a 20 months interval.

(A) Boxplots showing the chromatographic peak width (full width at half-maximum; FWHM) distributions of Hela peptides separated on the same PepMap column with different gradients in 2018 (red) and 2020 (blue; Orbitrap HF-X). (B) Boxplots showing the chromatographic peak width (full width at half-maximum; FWHM) distributions of Hela peptides separated on the same column using 60 min gradient between 2018 to 2020. Boxes cover 50% and whiskers represent 1.5 times the interquartile range of the data. (C) Overlaid pressure curves of Hela peptides separated on the same column between 2018 to 2020 using a 60 min gradient.

Figure 4. Application of the μLC-MS/MS system to the analysis of body fluid proteomes.

(A) Base peak chromatograms of crude plasma protein digest separated by micro-flow (upper panel, blue) and nano-flow (bottom panel, red) LC-MS/MS using 45 min measurement time. Selected peaks are annotated with peptide mass to charge (m/z) values. (B) Extracted ion chromatograms of one highly abundant peptide of m/z = 395.24. The x-axis represents a relative time scale to allow alignment of the two chromatograms. (C) Cumulative iBAQ (intensity based absolute quantification) intensity distribution of all quantified plasma and urine proteins following fractionation (48 fractions) by high-pH reversed phase chromatography and μLC-MS/MS analysis using 30 min gradient. Selected proteins of different abundance are marked by arrows. The inserted table shows how many proteins represent how much of the total iBAQ intensity.