Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures

Moskovets, Eugene; Ivanov, Alexander R.

doi:10.1007/s00216-016-9386-2

Cited by 6 publications

(8 citation statements)

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“…Besides the general positive effect on the location of minima in the van Deemter curve, the temperature has been shown to enhance the recovery of peptides in nano-LC–MS analyses, thus minimizing their carryover that applies particularly for hydrophobic peptides. , Elevated temperatures also improve peak shapes of analytes with protonatable moieties. This was originally explained by enhanced interaction kinetics of protonated analytes with deprotonated residual silanols, , although this might not be the only reason for the peak tailing of basic compounds using columns packed with silica-based stationary phases .…”

Section: Introductionmentioning

confidence: 99%

“…Besides the general positive effect on the location of minima in the van Deemter curve, the temperature has been shown to enhance the recovery of peptides in nano-LC−MS analyses, thus minimizing their carryover 13 that applies particularly for hydrophobic peptides. 14,15 Elevated temperatures also improve peak shapes of analytes with protonatable moieties.…”

Section: ■ Introductionmentioning

confidence: 99%

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Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC–MS Analyses

et al. 2020

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Elevated column temperature represents a simple means for improving chromatographic separation of peptides. Here, we demonstrated the advantages of the column temperature in peptide separation using state-of-the-art columns. More importantly, we also determined how temperature can impair proteomic bottom-up analyses. We found that an elevated temperature in combination with the acidic pH of the mobile phase induced in-column peptide hydrolysis with high specificity to Asp and accelerated five modification reactions of amino acids. The positive effects of temperature dominated in the 30 min long gradients since the column operated at 90 °C provided the largest number of identified peptides and proteins. However, the adverse effects of temperature on peptide integrity in longer liquid chromatography–mass spectrometry (LC–MS) analyses required its reduction to obtain optimum results. The largest number of peptides was identified using the column maintained at 75 °C in 60 min long gradients, at 60 °C in 120 min long gradients, and at 45 °C in 240 min long gradients. Our results indicate that no universal column temperature exists for bottom-up LC–MS analyses. Quite the contrary, the temperature setting must be selected rationally to exploit the full capabilities of the state-of-the-art mass spectrometers in proteomic LC–MS analyses, with the gradient time being a critical factor.

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Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC–MS Analyses

et al. 2020

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“…In terms of the peak shape for the QE+ analytical platform, the overall form differed from the three other platforms due to differences in chromatography (Figure ). Complex chromatography patterns due to internal prolines were detected. , …”

Section: Results and Discussionmentioning

confidence: 99%

Alternative LC–MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

et al. 2021

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Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides (GVPs) that contain single-amino-acid polymorphisms to infer the genotype of matching nonsynonymous single-nucleotide polymorphisms for the individual from whom the protein sample originated. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of GVPs increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data-dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data-independent acquisition and a variant of parallel reaction monitoring (PRM) on an Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring (MRM) on an Agilent 6495 triple quadrupole system. In our hands, average GVP detection from a selected panel of 24 GVPs increased from 6.5 ± 1.1 and 3.1 ± 0.8 using data-dependent and -independent acquisition to 9.5 ± 0.7 and 11.7 ± 1.7 using PRM and MRM (p < 0.05), respectively. PRM resulted in a 1.3-fold increase in detection sensitivity, and MRM resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median-estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For PRM and MRM, detected GVPs were validated by the inclusion of stable isotope-labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping and the establishment of validation methods for the evaluation of inferred genotypes.

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“…In terms of the peak shape for the QE+ analytical platform, the overall form differs from the three other platforms due to differences in chromatography (Figure 2). Complex chromatography patterns due to internal prolines were detected [52, 53].…”

Section: Resultsmentioning

confidence: 99%

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

Goecker¹,

Legg²,

Salemi

et al. 2021

Preprint

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Protein is a major component of all biological evidence. Proteomic genotyping is the use of genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of matching non-synonymous single nucleotide polymorphisms for the individual who originated the protein sample. This can be used to statistically associate an individual to evidence found at a crime scene. The utility of the inferred genotype increases as the detection of genetically variant peptides increases, which is the direct result of technology transfer to mass spectrometry platforms typically available. Digests of single (2 cm) human hair shafts from three European and two African subjects were analyzed using data dependent acquisition on a Q-Exactive Plus Hybrid Quadrupole-Orbitrap system, data independent acquisition and a variant of parallel reaction monitoring on a Orbitrap Fusion Lumos Tribrid system, and multiple reaction monitoring on an Agilent 6495 triple quadrupole system. In our hands, average genetically variant peptide detection from a selected 24 genetically variant peptide panel increased from 6.5 +/- 1.1 and 3.1 +/- 0.8 using data dependent and independent acquisition to 9.5 +/- 0.7 and 11.7 +/- 1.7 using parallel reaction monitoring and multiple reaction monitoring (p < 0.05). Parallel reaction monitoring resulted in a 1.3-fold increase in detection sensitivity, and multiple reaction monitoring resulted in a 1.6-fold increase in detection sensitivity. This increase in biomarker detection has a functional impact on the statistical association of a protein sample and an individual. Increased biomarker sensitivity, using Markov Chain Monte Carlo modeling, produced a median estimated random match probability of over 1 in 10 trillion from a single hair using targeted proteomics. For parallel reaction monitoring and multiple reaction monitoring, detected genetically variant peptides were validated by the inclusion of stable isotope labeled peptides in each sample, which served also as a detection trigger. This research accomplishes two aims: the demonstration of utility for alternative analytical platforms in proteomic genotyping, and the establishment of validation methods for the evaluation of inferred genotypes.

show abstract

Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures

Cited by 6 publications

References 75 publications

Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC–MS Analyses

Sense and Nonsense of Elevated Column Temperature in Proteomic Bottom-up LC–MS Analyses

Alternative LC–MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

Alternative LC-MS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts

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