Deep Proteome Profiling with Reduced Carryover Using Superficially Porous Microfabricated nanoLC Columns.

In the field of liquid chromatography-mass spectrometry (LC-MS)-based proteomics, increases in the sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of the data that can be generated do, however, also depend on the performance provided by nano-liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be important to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution. In the current contribution, we evaluate the use of the prototype generation 2 µPAC nanoLC columns, which use C18-functionalized superficially porous micropillars as a stationary phase. When compared to traditionally used fully porous silica stationary phases, more precursors could be characterized when performing single shot data-dependent LC-MS/MS analyses of a human cell line tryptic digest. Up to 30% more protein groups and 60% more unique peptides were identified for short gradients (10 min) and limited sample amounts (10-100 ng of cell lysate digest). With LC-MS gradient times of 10, 60, 120, and 180 min, respectively, we identified 2252, 6513, 7382, and 8174 protein groups with 25, 500, 1000, and 2000 ng of the sample loaded on the column. Reduction of sample carryover to the next run (up to 2 to 3%) and decreased levels of methionine oxidation (up to 3-fold) were identified as additional figures of merit. When analyzing a disuccinimidyl dibutyric urea-crosslinked synthetic library, 29 to 59 more unique crosslinked peptides could be identified at an experimentally validated false discovery rate of 1-2%.

Deep Single-Shot NanoLC-MS Proteome Profiling with a 1500 Bar UHPLC System, Long Fully Porous Columns, and HRAM MS.

This study demonstrates how the latest ultrahigh-performance liquid chromatography (UHPLC) technology can be combined with high-resolution accurate-mass (HRAM) mass spectrometry (MS) and long columns packed with fully porous particles to improve bottom-up proteomics analysis with nanoflow liquid chromatography-mass spectrometry (nanoLC-MS) methods. The increased back pressures from the UHPLC system enabled the use of 75 µm I.D. × 75 cm columns packed with 2 µm particles at a typical 300 nL/min flow rate as well as elevated and reduced flow rates. The constant pressure pump operation at 1500 bar reduced sample loading and column washing/equilibration stages and overall overhead time, which maximizes MS utilization time. The versatility of flow rate optimization to balance the sensitivity, throughput with sample loading amount, and capability of using longer gradients contributes to a greater number of peptide and protein identifications for single-shot bottom-up proteomics experiments. The routine proteome profiling and precise quantification of >7000 proteins with single-shot nanoLC-MS analysis open possibilities for large-scale discovery studies with a deep dive into the protein level alterations. Data are available via ProteomeXchange with identifier PXD035665.

Molecular markers for cervical cancer screening.

INTRODUCTION: Cervical cancer remains a significant healthcare problem, notably in low- to middle-income countries. While a negative test for hrHPV has a predictive value of more than 99.5%, its positive predictive value is less than 10% for CIN2+ stages. This makes the use of a so-called triage test indispensable for population-based screening to avoid referring women, that are ultimately at low risk of developing cervical cancer, to a gynecologist. This review will give an overview of tests that are based on epigenetic marker panels and protein markers. AREAS COVERED: There is a medical need for molecular markers with a better predictive value to discriminate hrHPV-positive women that are at risk of developing cervical cancer from those that are not. Areas covered are epigenetic and protein markers as well as health economic considerations in view of the fact that most cases of cervical cancer arise in low-to-middle-income countries. EXPERT OPINION: While there are biomarker assays based on changes at the nucleic acid (DNA methylation patterns, miRNAs) and at the protein level, they are not widely used in population screening. Combining nucleic acid-based and protein-based tests could improve the overall specificity for discriminating CIN2+ lesions that carry a low risk of progressing to cervical cancer within the screening interval from those that carry an elevated risk. The challenge is to reduce unnecessary referrals without an undesired increase in false-negative diagnoses resulting in cases of cervical cancer that could have been prevented. A further challenge is to develop tests for low-and middle-income countries, which is critical to reduce the worldwide burden of cervical cancer.

Online-2D NanoLC-MS for Crude Serum Proteome Profiling: Assessing Sample Preparation Impact on Proteome Composition.

Although current LC-MS technology permits scientists to efficiently screen clinical samples in translational research, e.g., steroids, biogenic amines, and even plasma or serum proteomes, in a daily routine, maintaining the balance between throughput and analytical depth is still a limiting factor. A typical approach to enhance the proteome depth is employing offline two-dimensional (2D) fractionation techniques before reversed-phase nanoLC-MS/MS analysis (1D-nanoLC-MS). These additional sample preparation steps usually require extensive sample manipulation, which could result in sample alteration and sample loss. Here, we present and compare 1D-nanoLC-MS with an automated online-2D high-pH RP × low pH RP separation method for deep proteome profiling using a nanoLC system coupled to a high-resolution accurate-mass mass spectrometer. The proof-of-principle study permitted the identification of ca. 500 proteins with ∼10,000 peptides in 15 enzymatically digested crude serum samples collected from healthy donors in 3 laboratories across Europe. The developed method identified 60% more peptides in comparison with conventional 1D nanoLC-MS/MS analysis with ca. 4 times lower throughput while retaining the quantitative information. Serum sample preparation related changes were revealed by applying unsupervised classification techniques and, therefore, must be taken into account while planning multicentric biomarker discovery and validation studies. Overall, this novel method reduces sample complexity and boosts the number of peptide and protein identifications without the need for extra sample handling procedures for samples equivalent to less than 1 µL of blood, which expands the space for potential biomarker discovery by looking deeper into the composition of biofluids.

Identification of 7 000-9 000 Proteins from Cell Lines and Tissues by Single-Shot Microflow LC-MS/MS.

A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 µL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of ∼9 000 proteins from 50 µg of protein digest from Arabidopsis roots, 7 500 from mouse thymus, and 7 300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by µLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.̀.