A Comprehensive Study of Gradient Conditions for Deep Proteome Discovery in a Complex Protein Matrix

Abstract

Bottom–up mass-spectrometry-based proteomics is a well-developed technology based on complex peptide mixtures from proteolytic cleavage of proteins and is widely applied in protein identification, characterization, and quantitation. A tims-ToF mass spectrometer is an excellent platform for bottom–up proteomics studies due to its rapid acquisition with high sensitivity. It remains challenging for bottom–up proteomics approaches to achieve 100% proteome coverage. Liquid chromatography (LC) is commonly used prior to mass spectrometry (MS) analysis to fractionate peptide mixtures, and the LC gradient can affect the peptide fractionation and proteome coverage. We investigated the effects of gradient type and time duration to find optimal gradient conditions. Five gradient types (linear, logarithm-like, exponent-like, stepwise, and step-linear), three different gradient lengths (22 min, 44 min, and 66 min), two sample loading amounts (100 ng and 200 ng), and two loading conditions (the use of trap column and no trap column) were studied. The effect of these chromatography variables on protein groups, peptides, and spectral counts using HeLa cell digests was explored. The results indicate that (1) a step-linear gradient performs best among the five gradient types studied; (2) the optimal gradient duration depends on protein sample loading amount; (3) the use of a trap column helps to enhance protein identification, especially low-abundance proteins; (4) MSFragger and PEAKS Studio have high similarity in protein group identification; (5) MSFragger identified more protein groups among the different gradient conditions compared to PEAKS Studio; and (6) combining results from both database search engines can expand identified protein groups by 9–11%.

Applications of tandem mass spectrometry (MS/MS) in antimicrobial peptides field: Current state and new applications

“…Faced with of the challenge of processing and analyzing high-throughput protein and peptide MS data, many comprehensive data analysis software platforms are available to assist us in the efficient and time-saving analysis and processing of such data. PEAKS Studio software (Waterloo, ON, Canada) is commonly used for data acquisition and analysis [ [145] , [175] , [176] , [177] , [178] ]. Additionally, software such as Proteome Discoverer [ 179 , 160 ], Mascot software [ 180 , 157 ], and ProteinLynx Global SERVER [ [148] , [181] , [182] ] are widely used in protein polypeptide analysis.…”

Proteomics—The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience

“…Regardless of the improvements in the speed and sensitivity of mass spectrometers, it is still impossible to analyse a whole proteome without some form of fractionation [ 174 , 175 ]. In BUP, many peptides will be essentially isobaric in their m/z value, leading to co-isolation for fragmentation, and thus complicated MS/MS spectra containing fragments from multiple peptides from different proteoforms [ 176 , 177 , 178 ].…”