Simple Peptide Quantification Approach for MS-Based Proteomics Quality Control

Abstract

Despite its growing popularity and use, bottom-up proteomics remains a complex analytical methodology. Its general workflow consists of three main steps: sample preparation, liquid chromatography coupled to tandem mass spectrometry (LC−MS/ MS), and computational data analysis. Quality assessment of the different steps and components of this workflow is instrumental to identify technical flaws and avoid loss of precious measurement time and sample material. However, assessment of the extent of sample losses along with the sample preparation protocol, in particular, after proteolytic digestion, is not yet routinely implemented because of the lack of an accurate and straightforward method to quantify peptides. Here, we report on the use of a microfluidic UV/ visible spectrophotometer to quantify MS-ready peptides directly in the MS-loading solvent, consuming only 2 μL of sample. We compared the performance of the microfluidic spectrophotometer with a standard device and determined the optimal sample amount for LC−MS/MS analysis on a Q Exactive HF mass spectrometer using a dilution series of a commercial K562 cell digest. A careful evaluation of selected LC and MS parameters allowed us to define 3 μg as an optimal peptide amount to be injected into this particular LC−MS/MS system. Finally, using tryptic digests from human HEK293T cells and showing that injecting equal peptide amounts, rather than approximate ones, result in less variable LC−MS/MS and protein quantification data. The obtained quality improvement together with easy implementation of the approach makes it possible to routinely quantify MS-ready peptides as a next step in daily proteomics quality control.

Whole-Genome Transformation Promotes tRNA Anticodon Suppressor Mutations under Stress

“…Cell aliquots, containing 2 mg of protein, were harvested after 10 h of fermentation, washed twice with PBS buffer, flash frozen in liquid nitrogen, and stored at −80°C. The samples were subjected to LC-MS/MS analysis of their whole proteomes and analyzed with validated methodology according to Maia et al ( 49 ) (VIB Proteomics Core Facility). Elution spectra after the LC runs indicated appropriate proteome coverage in all samples (see Fig.…”

“…Purified peptides were redissolved in 50 μl of loading solvent A (0.1% TFA in water/ACN [98:2, vol/vol]), and the peptide concentration determined on a Lunatic spectrophotometer (Unchained Labs, Ghent, Belgium) ( 49 ). Next, 2 μg of peptide material of each sample was injected for LC-MS/MS analysis in an Ultimate 3000 RSLC nano LC (Thermo Fisher Scientific, Bremen, Germany) connected in-line to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) equipped with a nanospray flex ion source (Thermo Fisher Scientific).…”

Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics

“…Purified peptides for shotgun analysis were redissolved in 20 µL solvent A (0.1% TFA in water/ACN (98:2, v/v) and peptide concentration was determined on a Lunatic spectrophotometer (Unchained Labs). 29 Two micrograms of each sample was injected for LC-MS/MS analysis on an UltiMate 3000 RSLCnano system in-line connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo) equipped with a pneu-Nimbus dual ion source (Phoenix S&T). Trapping was performed at 10 μL/min for 4 min in solvent A on a 20 mm trapping column (made in-house, 100 μm internal diameter (I.D.…”

Proteomics: A Tool to Study Platelet Function

“…With protein low-binding tubing material, the method could also be applicable for proteomic sample preparation. The additional controls, ensuring complete platelet lysis and digestion [ 117 , 118 ], minimal loss of peptide during sample preparation, and automation for the analysis of hundreds or thousands of samples should be established for high-throughput studies [ 119 ]. Finally, quality control systems of mass spectrometry-based proteomics data acquisition should be introduced in the labs dealing with platelets’ biomedical and translational applications, e.g., a cloud-based quality control system [ 120 ] or web-based applications [ 121 , 122 ].…”