Chapter Four · failure evidence
What Solid-Phase Peptide Extraction got wrong, from 21 dissertations
Solid-phase peptide extraction and purification methods frequently suffer from severe sample loss, unrecoverable matrix binding, and complete loss of detection sensitivity. Inappropriate buffer conditions, direct on-bead digestion, and complex multi-step purification cycles further cause analyte degradation, background contamination, and poor peptide recoveries. These records come from PhD theses at 10 institutions, 2021 to 2026. Each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Undetectable signal and low peptide recovery caused by omitted enrichment or reducing agents
Extracting endogenous peptides without targeted enrichment or omitting reducing agents during sample preparation led to concentrations below analytical detection limits and low peptide identification. Nanomaterial solid-phase extraction similarly failed to yield identifiable target peptides or open search hits during modified peptide profiling.
Tried and failed
nanomaterial solid-phase extraction for peptide enrichment applied to S-palmitoylated peptide profiling in cell lysates. Outcome: no signal. Reason: yielded almost no identifiable target peptides and zero hits in open search mode
Novel proteomics approaches to study S-acylation in biomolecular condensate regulation and disease · Imperial
Considered and rejected
Considered and rejected: Solid phase extraction / sample prep for mass spectrometry without any reducing agents was rejected due to resulting in very low peptide detection
Non-Lysine Acyl Modifications and Their Effects on Cellular Function · DukeSpace
Tried and failed
direct peptide extraction without enrichment applied to endogenous low-abundance tissue peptides. Outcome: no signal. Reason: endogenous peptide concentrations were below detection limits without targeted enrichment prior to mass spectrometry
Severe target peptide loss and unrecoverable binding during chromatographic separation
Chromatographic purification of dipeptide repeat proteins after solid-phase synthesis failed due to extreme aggregation and insolubility. Reversed-phase HPLC and anion exchange chromatography suffered from severe recovery loss, leaving target amyloidogenic peptides unrecovered or forcing a switch to precipitation.
Tried and failed
solid-phase peptide synthesis and chromatographic purification applied to hydrophobic dipeptide repeat proteins. Reason: extreme insolubility and aggregation prevented purification despite successful chemical synthesis
The Discovery and Chemical Synthesis of Peptides and Proteins that Cross Biological Barriers · MIT
Tried and failed
reversed-phase HPLC peptide purification applied to recombinantly expressed short amyloidogenic peptides. Outcome: no signal. Reason: cleaved peptide could not be detected or recovered from the chromatographic separation
Advances in Instrumentation for Dynamic Nuclear Polarization & Magic-Angle Spinning NMR · MIT
Considered and rejected
Considered and rejected: Rejected DEAE-C anion exchange chromatography for peptide purification due to substantial loss of peptide recovery, replacing it with cold 1:1 Et2O:hexane precipitation before HPLC.
Biosynthetic and Chemical Investigation of Lipid II-Binding Antimicrobials. · DukeSpace
High sample loss and operational complexity across multi-step purification cycles
Subjecting synthetic peptides to multi-step post-purification conjugation or sortagging resulted in low overall yields and complex workflows. Multi-labeling protocols for fluorosequencing and direct peptide copolymerization similarly caused substantial recovery losses across successive purification steps compared to simpler alternatives.
Lost to a baseline
Direct peptide monomer copolymerization yielded only 60% recovery of peptide monomer post-purification compared to near 100% conjugation efficiency in post-functionalization.
Engineering Intravenous Therapies for Trauma · ResearchWorks
Considered and rejected
Considered and rejected: Post-purification chemical conjugation via terminal cysteine or sortase transpeptidation (sortagging) of synthetic phosphorylated peptides was rejected due to complex multi-step purification and poor yields.
A materials-based approach for localized delivery of cancer immunotherapy · MIT
Considered and rejected
Considered and rejected: Rejected standard solution-phase multi-labeling for fluorosequencing due to sample losses during multiple purification cycles
Methods and technology development for bulk and single-molecule proteomics · UT Austin
Impaired peptide recovery and contamination from digestion buffers and on-bead processing
Direct on-bead proteolytic digestion of affinity eluates produced poorly reproducible recoveries alongside high background contamination from released streptavidin fragments. Modifying extraction buffers by adding salt to surfactants likewise substantially reduced overall peptide extraction efficiency.
Tried and failed
adding salt to surfactant extraction buffer applied to protein extraction from FFPE tissue. Outcome: worse than baseline. Reason: substantially reduced peptide recovery efficiency
Enabling Sensitive and Robust Characterization of Protein Therapeutics and Biomarkers in Biomatrices Through Novel LC-MS-Based Strategies · DSpace at SUNY Buffalo
Tried and failed
direct on-bead proteolytic digestion applied to streptavidin affinity purification eluates. Reason: poorly reproducible recovery and high background contamination from released streptavidin peptides
DISSECTING THE FUNCTION AND INTERACTOME OF PLEKHS1 IN PROSTATE TUMORIGENESIS · Cambridge
Left open by the authors
Problems the authors named and did not get to.
Left open
Optimize glycopeptide enrichment, purification, and ionization methods for mass spectrometry-based glycoproteomics analysis. Blocker: Requires wet lab access, chemical reagents, and physical mass spectrometry instruments for sample preparation and ionization
Left open
Remeasure extraction efficiency by extracting a diluted multianalyte standard using solid-phase extraction and LC-MS. Blocker: Requires access to a wet laboratory, multianalyte chemical standards, SPE cartridges, and a triple quadrupole LC-MS instrument
Tracking antibiotic pollution in urban waterways using solid-phase extraction and triple quadrupole LCMS · UT Austin
Left open
Extend the SMART peptide purification method to complex peptide mixtures and pilot large-scale chromatography manufacturing runs. Blocker: Requires a wet chemistry lab, preparative chromatography equipment, and peptide synthesis/purification materials.
Fundamentals of peptide adsorption in reversed-phase liquid chromatography · Imperial
Left open
Develop a solid-phase aniline derivatization protocol to combine chemical derivatization and extraction into a single step for dissolved organic matter analysis. Blocker: Requires a wet chemistry laboratory, solid-phase extraction materials, and UHPLC-ESI-MS/MS instrumentation.
Molecular characterization of microbial interactions with labile dissolved organic matter · MIT
Left open
Optimize protein purification conditions for SLC25A51 to yield sufficient protein for proteoliposome reconstitution and structural microscopy. Blocker: Requires a wet biochemistry lab, physical reagents, and specialized structural microscopy equipment
Labeled nicotinamide adenine dinucleotide reveals the mechanism of mitochondrial NAD⁺ replenishment · UT Austin
Left open
Compare the analytical performance of the QuEChERS extraction method against solid-phase extraction (SPE) for wastewater surveillance samples. Blocker: Requires a wet chemistry laboratory, chemical reagents, wastewater samples, and analytical instruments (e.g., LC-MS).
Applications of Wastewater Surveillance in a Small-Scale Wastewater System · DalSpace
Left open
Develop molecularly imprinted polymer solid-phase extraction (MIP-SPE) methods to enrich polyphosphonates for compound-specific isotope analysis at low microgram-per-liter concentrations. Blocker: Requires chemical synthesis of molecularly imprinted polymers and physical wet-lab analytical equipment (LC-IRMS)
Manganese–driven oxidation of aminopolyphosphonates studied by compound–specific carbon isotope analysis · Publikationssystem UB Tuebingen
Left open
Develop an extraction and secondary chromatographic method using polar sorbents or anion exchange resins to retain short-chain PFAS analytes. Blocker: Requires an analytical chemistry wet lab, chromatography equipment, and chemical standards.
Evaluating "Forever Chemicals" from Soup to Nuts: Sample Preparation, Targeted Analysis, and Suspect Screening for the Characterization of Per- and Polyfluoroalkyl Substances (PFAS · DSpace at SUNY Buffalo
Left open
Measure anion exchange sorbent affinity for phosphorothioate oligonucleotides, phosphorylated peptides, lipids, and proteins using coated planar nitinol extraction devices. Blocker: Requires a wet chemistry lab, functionalized nitinol substrates, sorbents, and analytical instruments (e.g., LC-MS, fluorescence spectrometers).
Improving bioanalytical methods for nucleic acids with chemically tuned separations · Iowa State
Left open
Conjugate anionic polymers PP50 and PLP-NDA18 with amidated ova30 peptide to overcome electrostatic charge repulsion during delivery. Blocker: Requires chemical synthesis, purification, and wet-lab delivery assay facilities
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