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Limited impact of column chemistry and length on proteome coverage under high-speed DIA

ABSTRACT: The evolution of mass spectrometry-based proteomics has been driven by continuous advances in instrumentation, sample preparation, and data acquisition strategies. While chromatographic separation has historically been considered a critical bottleneck in achieving comprehensive proteome coverage, recent developments in ultra-fast mass spectrometry acquisition fundamentally challenge this paradigm. We investigated whether traditional chromatographic optimization principles established during the early era of proteomics remain essential in contemporary workflows. Using five distinct stationary phases, C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluated proteome identification performance using state-of-the-art data-independent acquisition on the Orbitrap Astral mass spectrometer with HeLa tryptic digests. Despite substantial chromatographic differences in selectivity and peak characteristics that would have profoundly influenced analytical outcomes in earlier instrumentation generations, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor and approximately 9,000 protein group identifications, regardless of column length variations. While distinct selectivity fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in bulk identification depth under high-speed acquisition conditions exceeding 200 Hz. From these findings we conclude that the analytical bottleneck has fundamentally shifted from chromatographic resolution to mass spectrometric sampling efficiency, where comprehensive peptide identification is now achieved through advanced spectral deconvolution rather than physical separation alone. This paradigmatic shift suggests that method development priorities in modern proteomics should evolve beyond traditional separation optimization to emphasize operational robustness, analytical throughput, and systematic reproducibility for routine applications.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Mario Oroshi

LAB HEAD: Dr. Johannes Bruno Müller-Reif

PROVIDER: PXD067710 | Pride | 2026-06-22

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	DIANN_reports.zip	Other
	Raw_files_ExsilMono100_C18_1p35um.zip	Other
	Raw_files_ReproSilSaphir100_C18_1p3um.zip	Other
	Raw_files_Reprospher100_C8_1p9um.zip	Other
	Raw_files_Reprospher100_PhenylHexyl_1p8um.zip	Other

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Project description:The evolution of mass spectrometry-based proteomics has been driven by continuous advances in instrumentation, sample preparation, and data acquisition strategies. While chromatographic separation has historically been considered a critical bottleneck in achieving comprehensive proteome coverage, recent developments in ultra-fast mass spectrometry acquisition fundamentally challenge this paradigm. We investigated whether traditional chromatographic optimization principles established during the early era of proteomics remain essential in contemporary workflows. Using five distinct stationary phases, C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluated proteome identification performance using state-of-the-art data-independent acquisition on the Orbitrap Astral mass spectrometer with HeLa tryptic digests. Despite substantial chromatographic differences in selectivity and peak characteristics that would have profoundly influenced analytical outcomes in earlier instrumentation generations, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor and approximately 9,000 protein group identifications, regardless of column length variations. While distinct selectivity fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in bulk identification depth under high-speed acquisition conditions exceeding 200 Hz. From these findings we conclude that the analytical bottleneck has fundamentally shifted from chromatographic resolution to mass spectrometric sampling efficiency, where comprehensive peptide identification is now achieved through advanced spectral deconvolution rather than physical separation alone. This paradigmatic shift suggests that method development priorities in modern proteomics should evolve beyond traditional separation optimization to emphasize operational robustness, analytical throughput, and systematic reproducibility for routine applications.

Project description:Differential proteome profiling of IBD serum exosomes to find corresponding regulatory protein cargoes of serum exosomes in IBD progress. Exosomal proteins were extracted using SDT buffer lysis (4% SDS, 100mM Tris HCl, 1mM DTT, pH7.6), and the protein concentration was determined using the BCA protein quantification kit (Bio Rad, USA). The protein was digested with pancreatin and processed by ultrafiltration assisted sample preparation (FASP) method. The peptide segments obtained after treatment were desalinated using a C18 chromatographic column, concentrated by vacuum centrifugation, and then dissolved in 40 µL 0.1% formic acid. Identification and analysis were conducted using liquid chromatography tandem mass spectrometry (LC-MS/MS) technology. Each peptide sample was separated using a nanoliter flow rate high-performance liquid chromatography system, with mobile phase buffer A containing 0.1% formic acid aqueous solution and B containing 0.1% formic acid acetonitrile aqueous solution (84% acetonitrile). Using a 95% A-liquid equilibrium chromatographic column, the sample is loaded into a C18 chromatographic column using an automatic sampler, and separated by a C18-A2 analytical column at a flow rate of 300 nL/min. After chromatographic separation, the sample was subjected to mass spectrometry analysis using the timsTOF Pro mass spectrometer. The detection method is positive ions, the ion source voltage is set to 1.5kV, and TOF is used for detection and analysis in both mass spectrometry and tandem mass spectrometry. The scanning range of the mass spectrometry is set to 100-1700m/z. The data collection mode adopts the Parallel Accumulation Serial Fragmentation (PASEF) mode. After collection, the mother ion is collected in 10 PASEF modes, with a cycle window time of 1.17 seconds and a secondary mass spectrometry with a charge number in the range of 0-5, The dynamic exclusion time of tandem mass spectrometry scanning is set to 24 seconds to avoid repeated scanning of parent ions and generate original mass spectrometry detection data. The mass spectrometry raw data was identified and quantitatively analyzed using the LFQ (Label Free Quantification) algorithm using MaxQuant software (version 1.6.14).

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Limited impact of column chemistry and length on proteome coverage under high-speed DIA

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