Phosphopeptide Ionization via Low pH DESI

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Solution Overview

Problem

Phosphopeptide signals are severely suppressed during mass spectrometry analysis due to ionization methods like electrospray ionization, requiring laborious purification steps, and there is a need for a direct and rapid method to analyze phosphopeptides in mixtures without damaging the mass spectrometer.

Innovation Solution

The method involves acidifying phosphopeptides with a strong acid like hydrochloric acid to suppress deprotonization, followed by desorption electrospray ionization (DESI) mass spectrometry, which dilutes the acid and allows for effective ionization and analysis without instrument damage.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional electrospray ionization is used to ionize phosphoprotein digest, then ionization occurs, but phosphopeptide signal is severely suppressed by non-phosphorylated peptides

Engineering Contradiction:

Improvephosphopeptide signal detectionVSAvoidion suppression by non-phosphorylated peptides

Core Design Contradiction:

Measurement precisionVSObject-generated harmful factors

Solution Approach 1:

The invention changes the pH parameter of the ionization environment to extremely low values (pH < 2, preferably pH < 1) using strong acids like HCl or TFA. This parameter change fundamentally alters the ionization behavior, allowing phosphopeptides to be protonated and detected in positive ion mode while non-phosphorylated peptides remain suppressed, thereby resolving the ion suppression problem

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

Instead of using the conventional approach of enriching phosphopeptides before analysis, the invention inverts the strategy by using low pH conditions that naturally suppress non-phosphorylated peptides and enhance phosphopeptide signals directly in the mass spectrometer, eliminating the need for preliminary purification steps

Inventive Principle:

Principle #13The other way round (Inversion)

2Measurement precision

If strong acid is added to suppress deprotonization of phosphate groups, then phosphopeptide ionization is enhanced, but mass spectrometer may be damaged by strong acid

Engineering Contradiction:

Improvephosphopeptide ionization efficiencyVSAvoidmass spectrometer damage from strong acid

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The invention introduces a mediator substance (such as isopropanol, acetonitrile, or other organic modifiers) that is mixed with the strong acid solution. This mediator protects the mass spectrometer components from direct exposure to concentrated strong acid while still allowing the low pH conditions to be maintained for effective phosphopeptide ionization, thus resolving the contradiction between ionization efficiency and instrument safety

Inventive Principle:

Principle #24Intermediary (Mediator)

3Measurement precision

If preliminary purification of phosphopeptides is performed using antibodies or affinity chromatography, then phosphopeptide enrichment is achieved, but analysis time is increased

Engineering Contradiction:

Improvephosphopeptide enrichmentVSAvoidpurification time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The invention extracts and eliminates the time-consuming purification step entirely by using low pH direct infusion mass spectrometry. The strong acid conditions naturally suppress non-phosphorylated peptides and enhance phosphopeptide signals, allowing direct analysis of phosphoprotein digests without requiring antibody-based enrichment or affinity chromatography, thereby saving significant analysis time while maintaining detection precision

Inventive Principle:

Principle #2Taking out (Extraction)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach prevents signal suppression, enables 100% coverage of phosphorylated peptides with minimal sample quantity, and enhances charge states for tandem MS analysis, reducing analysis time and cost by eliminating the need for preliminary purification.

Implementation Method 1

The phosphopeptide can be acidified with a strong acid, such as hydrochloric acid, which suppresses the deprotonization of the peptide phosphate groups in solution

Methodology Applied

Scientific EffectAcid-base reaction: Chemical Bonding

Implementation Method 2

Sample ionization by DESI occurs via the interactions with charged microdroplets generated in a pneumatically assisted electrospray of an appropriate solvent

Methodology Applied

Scientific EffectElectrospray ionization: Electrohydrodynamics

Implementation Method 3

the ionizing electrospray effectively dilutes the acidified phosphopeptide, allowing it to be analyzed using MS without damaging the mass spectrometer

Methodology Applied

Scientific EffectDilution:

Implementation Method 4

DESI provides direct ionization of analytes with little or no sample preparation... allowing it to be analyzed using MS

Methodology Applied

Scientific EffectMass spectrometry:

Data Source

PatentUS9977028B2Ionization of chemicals in mixture at low pH by ambient ionization/mass spectrometry

Publication Date: 2018.05.22 OHIO UNIV

AI summary

A mass spectrometry-based method for analyzing an acidic organic target compound includes directing a charged solvent (44) toward a pre-acidified sample (12) comprising the target compound, to thereby ionize the pre-acidified sample (12). The method further includes directing the ionized pre-acidified sample (54) to a mass spectrometer (18), the mass spectrometer (18) being configured to identify and quantify the target compound.