Reduce Ion Suppression Research Articles

Photoinduced Online Enrichment-Deglycosylation of Glycolipids for Enhancing Lipid Coverage and Identification in Single-Cell Mass Spectrometry.

Single-cell lipidomics provides important information for molecular mechanisms of living processes and diseases at the individual cell level. However, single-cell lipidomic mass spectrometry (MS) techniques suffer from low lipid coverage and incomplete structural elucidation, especially for poorly ionizable glycosphingolipids (GSLs). Herein, a photoinduced enrichment-deglycosylation method of GSLs was developed and introduced into an ambient liquid extraction MS system for enhancing detection coverage and identification accuracy of GSLs in single-cell MS. GSL standards were selectively adsorbed on TiO2 in ammonia-added protic solvents. Under UV irradiation, the adsorbed GSLs would lose one hexosyl group (deglycosylation), and the products (>70% conversion efficiency) were desorbed from TiO2. By coating porous TiO2 into the capillary of the ambient liquid extraction MS system, online adsorption of GSLs and their separation from high-abundance phospholipids were achieved, largely reducing ion suppression. By UV irradiation, captured GSLs were rapidly deglycosylated and photodesorbed from TiO2 coating without solvent switching, resulting in 6-fold enrichment. With the new method, the detection coverage of GSLs was enhanced 9-fold without losing other lipidomes, compared with the conventional method. Moreover, deglycosylated GSLs from photodesorption had more MS/MS fragments than intact GSLs, facilitating detailed fatty acyl and sphingosine chain elucidation. Seven deglycosylated GSL peaks were identified with the confirmed hydroxyl group location in the fatty acyl chain, while only 1 was identified for intact GSL. The new method was applied to the single-cell lipidomics study of two types of nerve cells. Totally, 31 lipids including 11 GSLs were identified in a single cell, and 5 hexosylceramides were found significantly altered after neuron injury.

Crown ether dopant to reduce ion suppression and improve detection in the liquid microjunction surface sampling probe.

Sodium and potassium are required in agar media for the growth of some microorganisms (e.g., marine bacteria). However, alkali cations are a significant source of contamination for mass spectrometry causing ion suppression and adduct formation. Conventionally, salts can be removed before mass spectrometric analysis with appropriate and often lengthy sample preparation. The direct mass spectrometric sampling of bacterial colonies grown on agar media seeks to minimize or eliminate sample preparation to improve workflow. However, this may exacerbate ion suppression and contamination since these metal cations will degrade spectral quality and limit the rapid profiling of microbial metabolites. Different approaches are needed to sequester sodium and potassium ions to minimize unwanted background interferences. Herein, we use crown ethers (CEs) in combination with a liquid microjunction surface sampling probe (LMJ-SSP) to directly sample the surface of the bacterial colonies from two marine bacteria species (Pseudoalteromonas rubra DSM6842 and Pseudoalteromonas tunicata DSM 14096). CEs (e.g., 18-crown-6 or 15-crown-5) are added to the carrier solvent of the LMJ-SSP, the chemical noise is reduced, and spectra are easier to interpret. The liquid microjunction formed at the tip of LMJ-SSP was used to directly touch bacterial colonies on agar. The carrier solvent was either methanol (100%) or methanol: H2O (50:49.9%) with or without 0.01% CEs. Information-theoretic measures are employed to investigate qualitative changes between spectra before and after adding CEs. Our work demonstrates the capability of CEs to reduce background interferences within the direct profiling of bacterial colonies from agar plates. The data obtained from both P. rubra DSM6842 and P. tunicata DSM 14096 show that CEs can be used to mitigate the salty background and improve compound detection. Our approach can be implemented in natural product discovery using LMJ-SSP to allow fast and accurate detection of interesting/novel compounds.

Nitro Indole Derivatives as Novel Dual-Polarity Matrices for MALDI Mass Spectrometry and Imaging with Broad Applications.

A new matrix framework is presented in this study for the improved ionization efficiency of complex mixtures by matrix-assisted laser desorption ionization (MALDI) mass spectrometry/imaging. Five nitro indole (NI) derivatives [3-methyl-4-nitro-1H-indole (3,4-MNI), 3-methyl-6-nitro-1H-indole (3,6-MNI), 2,3-dimethyl-4-nitro-1H-indole (2,3,4-DMNI), 2,3-dimethyl-6-nitro-1H-indole (2,3,6-DMNI), and 4-nitro-1H-indole (4-NI)] were synthesized and shown to produce both positive and negative ions with a broad class of analytes as MALDI matrices. NI matrices were compared to several common matrices, such as 2,5-dihydroxybenzoic acid (DHB), alpha-cyano-4-hydroxylcinnamic acid (CHCA), sinapinic acid (SA), 1,5-diaminonaphthelene (1,5-DAN), and 9-aminoacridine (9-AA), for the analysis of lipid, peptide, protein, glycan, and perfluorooctanesulfonic acid (PFOS) compounds. 3,4-MNI demonstrated the best performance among the NI matrices. This matrix resulted in reduced ion suppression and better detection sensitivity for complex mixtures, for example, egg lipids/milk proteins/PFOS in tap water, while 2,3,6-DMNI was the best matrix for blueberry tissue imaging. Several important aspects of this work are reported: (1) dual-polarity ion production with NI matrices and complex mixtures; (2) quantitative analysis of PFOS with a LOQ of 0.5 ppb in tap water and 0.05 ppb in MQ water (without solid phase extraction enrichment), with accuracy and precision within 5%; (3) MALDI imaging with 2,3,6-DMNI as a matrix for plant metabolite/lipid identification with ionization enhancement in the negative ion mode m/z 600-900 region; and (4) development of a thin film deposition under/above tissue method for MALDI imaging with a vacuum sublimation matrix on a high-vacuum MALDI instrument.

Lewis Acidic Metal-Organic Framework Assisted Ambient Liquid Extraction Mass Spectrometry Imaging for Enhancing the Coverage of Poorly Ionizable Lipids in Brain Tissue.

The spatial distribution of lipidomes in tissues is of great importance in studies of living processes, diseases, and therapies. Mass spectrometry imaging (MSI) has become a critical technique for spatial lipidomics. However, MSI of low-abundance or poorly ionizable lipids is still challenging because of the ion suppression from high-abundance lipids. Here, a metal-organic framework (MOF) Zr6O4(OH)4(1,3,5-Tris(4-carboxyphenyl) benzene)2(triflate)6(Zr6OTf-BTB) was prepared and used for selective on-tissue adsorption of phospholipids to reduce ion suppression from them to poorly ionizable lipids. The results show that Zr6OTf-BTB with strong Lewis acidic sites and a large specific surface area (647.9 m2·g-1) could selectively adsorb phospholipids under 1% FA-MeOH. Adsorption efficiencies of phospholipids are 88.4-144.9 times higher than those of other neutral lipids. Moreover, the adsorption capacity and the adsorption kinetic rate constant of the new material to phospholipids are higher than those of Zr6-BTB (242.72 vs 73.96 mg·g-1, 0.0442 vs 0.0220 g·mg-1·min-1). A Zr6OTf-BTB sheet was prepared by a lamination technique for on-tissue phospholipid adsorption from brain tissue. Then, the tissue section on the Zr6OTf-BTB sheet was directly imaged via ambient liquid extraction-MSI with 1% FA-MeOH as the sampling solvent. The results showed that phospholipids could be 100% removed directly on tissue, and the detection coverage of the Zr6OTf-BTB-enhanced MSI method to ceramides (Cers) and hexosylceramides (HexCers) was increased by 5-26 times compared with direct tissue MSI (26 vs 1 and 17 vs 3). The new method provides an efficient and convenient way to eliminate the ion suppression from phospholipids in MSI, largely improving the detection coverage of low-abundance and poorly ionizable lipids.

Photocatalytic degradation-based ambient mass spectrometry imaging for enhancing detection coverage of poorly-ionizable lipidomes

Localization of lipidomes and tracking their spatial changes in tissues by mass spectrometry imaging (MSI) plays an important role in unveiling the mechanisms of living processes, diseases and therapeutic treatments. However, it is always challenging to achieve direct MSI of poorly-ionizable lipids, such as glycolipids and glycerolipids, due to the strong ion suppression and isobaric peaks interference from high-abundance phosphatidylcholines (PCs) in tissues. Here we developed a photocatalytic degradation-based ambient liquid extraction MSI method to largely enhance the detection coverage of poorly-ionizable lipids by rapid online removal of PCs in MSI. Phospholipids were found to be selectively photodegraded on TiO2 surface in acidic conditions in the presence of water under UV irradiation, while other poorly-ionizable lipids remained. Sulfate ion could largely improve the degradation efficiencies. Anatase nanoparticles-embedded TiO2 monolith was in-situ synthesized in the capillary of ambient liquid extraction system, and rapid online photodegradation of PCs was achieved during MSI with efficiency >80 %, largely reducing ion suppression. The pathway analysis showed that PC was oxidatively degraded starting from hydroxylation of C=C bonds. With intense UV irradiation, PCs were completely degraded into small molecules<200 Da without interference on the detection of endogenous lipids. With the new MSI method, detection coverage to cerebrosides, ceramides and diglycerides was enhanced by 2–9 times comparing with traditional MSI. Clearer localizations were observed for poorly-ionizable lipids via the new method than traditional method. Thus, this work provided a complementary MSI method for traditional MSI to address the issues on direct imaging of poorly ionizable lipids in ambient conditions.

Pros and Cons of Separation, Fractionation and Cleanup for Enhancement of the Quantitative Analysis of Bitumen-Derived Organics in Process-Affected Waters—A Review

Oil sands process-affected water (OSPW) contains a diverse mixture of inorganic and organic compounds. Naphthenic acids (NAs) are a subset of the organic naphthenic acid fraction compounds (NAFCs) and are a major contributor of toxicity to aquatic species. Thousands of unique chemical formulae are measured in OSPW by accurate mass spectrometry and high-resolution mass spectrometry (MS) analysis of NAFCs. As no commercial reference standard is available to cover the range of compounds present in NAFCs, quantitation may best be referred to as “semi-quantitative” and is based on the responses of one or more model compounds. Negative mode electrospray ionization (ESI-) is often used for NAFC measurement but is prone to ion suppression in complex matrices. This review discusses aspects of off-line sample preparation techniques and liquid chromatography (LC) separations to help reduce ion suppression effects and improve the comparability of both inter-laboratory and intra-laboratory results. Alternative approaches to the analytical parameters discussed include extraction solvents, salt content of samples, extraction pH, off-line sample cleanup, on-line LC chromatography, calibration standards, MS ionization modes, NAFC compound classes, MS mass resolution, and the use of internal standards.

Boronate affinity paper spray mass spectrometry for determination of elevated levels of catecholamines in urine

The analysis of catecholamines, such as dopamine, epinephrine and norepinephrine in urine can be used in the diagnosis of certain pathologies, such as hormone-producing tumors. Here, a fast and simple quantitative boronate affinity paper spray tandem mass spectrometric (PS-MS/MS) method is established, which can improve selectivity and reduce ion suppression without needing any instrumental chromatography. We use here the property of boronic acids, which can selectively bind ortho-diol-containing compounds under alkaline conditions. Paper tip modification and catechol enrichment protocols were developed to selectively bind, clean up and subsequently desorb such catecholamines. Standard catecholamine solutions, as well as human urine samples were analyzed with the PS-MS(/MS) method, which is fast, cheap and easy-to-operate compared to HPLC-MS/MS. Despite its high simplicity, boronate affinity PS-MS/MS exhibits good performance compared to HPLC-MS/MS in human urine analysis in terms of precision (2.1%–7.2% vs. 1.1%–2.9%) and accuracy (−10.2%–9.3% vs. −4.8%–5.1%), and a physiologically relevant limit of detection (0.027–0.12 μg mL−1). The boronate affinity PS-MS/MS clearly achieved the detection limits that would allow the fast analysis of urine samples for clinical purposes, such as screening for pheochromocytoma (exceeding 0.5 μg mL−1).

Introducing Nafion for In Situ Desalting and Biofluid Profiling in Spray Mass Spectrometry.

We introduce Nafion into the ambient ionization technique of spray mass spectrometry to serve for in situ desalting and direct analysis of biological fluids. Nafion was coated onto the surface of the triangular spray tip as the cation exchange material. Because the sulfonic group from the Nafion membrane effectively exchanges their carried protons with inorganic salt ions (e.g., Na+ and K+), the analyte’s ionization efficiency can be significantly enhanced by reducing ion suppression. The desalting efficiency can reach 90% and the maximum tolerance of the absolute salt amount reaches 100 μmol. The mass spectral profile can also be simplified by removing the multiple adducted ion types from small-molecule drugs and metabolites ([M + Na]+ and [M + K]+), or multiply charged ions formed by proteins ([M + nNa]n+ and [M + nK]n+). Thus, the Nafion coating makes less ambiguous data interpretation collected from spray mass spectrometry for qualitative profiling or quantitative measurement of a target analyte.

Multidimensional Mass Spectrometry Assisted Metallo-Supramolecular Chemistry

Multidimensional Mass Spectrometry Assisted Metallo-Supramolecular Chemistry

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

The critical role of mobile phase pH in the performance of oligonucleotide ion-pair liquid chromatography-mass spectrometry methods.

Current applications of capillary electrophoresis-mass spectrometry for the analysis of biologically important analytes in urine (2017 to mid-2021): A review.

Capillary electrophoresis coupled online with mass detection is a modern tool for analyzing wide ranges of compounds in complex samples, including urine. Capillary electrophoresis with mass spectrometry allows the separation and identification of various analytes spanning from small ions to high molecular weight protein complexes. Similarly to the much more common liquid chromatography‐mass spectrometry techniques, the capillary electrophoresis separation reduces the complexity of the mixture of analytes entering the mass spectrometer resulting in reduced ion suppression and a more straightforward interpretation of the mass spectrometry data. This review summarizes capillary electrophoresis with mass spectrometry studies published between the years 2017 and 2021, aiming at the determination of various compounds excreted in urine. The properties of the urine, including its diagnostical and analytical features and chemical composition, are also discussed including general protocols for the urine sample preparation. The mechanism of the electrophoretic separation and the instrumentation for capillary electrophoresis with mass spectrometry coupling is also included. This review shows the potential of the capillary electrophoresis with mass spectrometry technique for the analyses of different kinds of analytes in a complex biological matrix. The discussed applications are divided into two main groups (capillary electrophoresis with mass spectrometry for the determination of drugs and drugs of abuse in urine and capillary electrophoresis with mass spectrometry for the studies of urinary metabolome).

Determination of acrylamide in gingerbread and other food samples by HILIC-MS/MS: A dilute-and-shoot method

This paper describes a hydrophilic interaction liquid chromatography tandem mass spectrometric (HILIC-MS/MS) method for determining acrylamide in food. The method primary optimised for gingerbread samples that have high sugar contents. A new sample preparation process was optimized using an experiment design based on a central composition design (CCD). A mixture of acidified aqueous acetonitrile was established as a suitable extraction medium. The extracts were further diluted and separation of acrylamide on TSKgel Amide-80 HILIC column was carried out within 8 min. The method was validated using naturally contaminated quality check (QC) as well as spiked samples. The developed method showed acceptable accuracy (101% − 105%) and precision (2.9% − 7.6%). The limit of quantification was 20 µg/kg. The method was also tested by analysing acrylamide in other food samples (bread, roasted coffee, instant coffee, cappuccino powder and fried potato). The acrylamide concentrations found in samples were between 20 µg/kg and 667 µg/kg, which were lower than the benchmark levels set by the European Union (EU). The main advantage of the newly developed method over the standard methods included the easier sample preparation and faster analysis with reduced ion suppression.

Supercritical fluid chromatography coupled to tandem mass spectrometry for the analysis of pesticide residues in dried spices. Benefits and drawbacks

This study describes the high sensitivity and the reduced ion suppression and matrix effect that can be achieved by supercritical fluid chromatography (SFC) in the analysis of dried spices as complex matrices. Samples selected for the evaluation were cayenne and black pepper, which are representative of complex dried spices. To carry out the evaluation of this technique, blank samples were fortified with a solution containing 162 pesticides at two concentration levels (50 and 200 μg kg−1). During the modified QuEChERS method, EMR sorbent was used as dispersive SPE in the clean-up step. The samples were analysed by supercritical fluid chromatography coupled to tandem mass spectrometry. The validation parameters studied were recovery, inter and intraday precision, linearity and matrix effect. Recoveries for the majority of compounds were in the 70–120% range recommended by DG-SANTE guidelines and showed a precision lower than 20% in terms of RSD. Matrix effect was low (0–20% signal suppression) for 132 pesticides in cayenne and 91 in black pepper. The method was used to analyse 47 real samples of spices from different countries. A high number of these samples presented one or more pesticides (81%). Some of these detected pesticides are not approved in the European Union and, in many cases, the positive findings exposed concentrations that exceed the EU MRLs.

Sensitive and Selective Amino Acid Profiling of Minute Tissue Amounts by HPLC/Electrospray Negative Tandem Mass Spectrometry Using 9-Fluorenylmethoxycarbonyl (Fmoc-Cl) Derivatization.

A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50-100 samples a day. In addition to a detailed description of the method, some representative examples are presented.

Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism

AbstractWhile metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p‐nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota‐derived metabolites.

Chemoselective Probe Containing a Unique Bioorthogonal Cleavage Site for Investigation of Gut Microbiota Metabolism.

While metabolites derived from gut microbiota metabolism have been linked to disease development in the human host, the chemical tools required for their detailed analysis and the discovery of biomarkers are limited. A unique and multifunctional chemical probe for mass spectrometric analysis, which contains p-nitrocinnamyloxycarbonyl as a new bioorthogonal cleavage site has been designed and synthesized. Coupled to magnetic beads, this chemical probe allows for straightforward extraction of metabolites from human samples and release under mild conditions. This isolation from the sample matrix results in significantly reduced ion suppression, an increased mass spectrometric sensitivity, and facilitates the detection of metabolites in femtomole quantities. The chemoselective probe was applied to the analysis of human fecal samples, resulting in the discovery of four metabolites previously unreported in this sample type and confirmation of the presence of medically relevant gut microbiota-derived metabolites.

Non-target analysis of household dust and laundry dryer lint using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

Household dust and laundry dryer lint are important indoor environmental matrices that may have notable health effects on humans due to chronic exposure. However, due to the sample complexity the studies conducted on these sample matrices until now were almost exclusively on the basis of target analysis. In this study, comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry (LC × LC-ToF MS) was applied, to enable non-target analysis of household dust as well as laundry dryer lint for the first time. The higher peak capacity and good orthogonality of LC × LC, together with reduced ion suppression in the MS enabled rapid identification of environmental contaminants in these complex sample matrices. A number of environmental contaminants were tentatively identified based on their accurate masses and isotopic patterns, including plasticizers, flame retardants, pesticides, drug metabolites, etc. The identity of seven compounds: tris(2-butoxyethyl) phosphate, tris(2-chloropropyl) phosphate, n-benzyl butyl phthalate, dibutyl phthalate, tributyl phosphate, triethyl phosphate and N, N-diethyl-meta-toluamide was confirmed using two-dimensional retention alignment and their concentrations in the samples were semi-quantitatively determined.

Modified HPLC-ESI-MS Method for Glycated Hemoglobin Quantification Based on the IFCC Reference Measurement Procedure and Its Application for Quantitative Analyses in Clinical Laboratories of China.

The level of glycated hemoglobin (HbA1c ) has been recognized as an important indicator of long-term glycemic control. However, the HbA1c measurement is not currently included as a diagnostic determinant in China. Current study aims to assess a candidate modified International Federation of Clinical Chemistry reference method for the forthcoming standardization of HbA1c measurements in China. The HbA1c concentration was measured using a modified high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method. The modified method replaces the propylcyanide column with a C18 reversed-phase column, which has a lower cost and is more commonly used in China, and uses 0.1% (26.5 mmol/l) formic acid instead of trifluoroacetic acid. Moreover, in order to minimize matrix interference and reduce the running time, a solid-phase extraction was employed. The discrepancies between HbA1c measurements using conventional methods and the HPLC-ESI-MS method were clarified in clinical samples from healthy people and diabetic patients. Corresponding samples were distributed to 89 hospitals in Beijing for external quality assessment. The linearity, reliability, and accuracy of the modified HPLC-ESI-MS method with a shortened running time of 6 min were successfully validated. Out of 89 hospitals evaluated, the relative biases of HbA1c concentrations were < 8% for 74 hospitals and < 5% for 60 hospitals. Compared with other conventional methods, HbA1c concentrations determined by HPLC methods were similar to the values obtained from the current HPLC-ESI-MS method. The HPLC-ESI-MS method represents an improvement over existing methods and provides a simple, stable, and rapid HbA1c measurement with strong signal intensities and reduced ion suppression.

Phospholipid removal combined with a semi-automated 96-well SPE application for determination of budesonide in human plasma with LC–MS/MS

A semi-automated method for quantification of budesonide in human plasma was developed, validated, and applied for high-volume analysis of samples in connection with a pharmacokinetic study. Protein and phospholipid removal was performed using an Ostro 96-well filter plate and subsequently combined with C18 solid-phase extraction on a Hamilton Microlab STARlet automation robot. The final extracts were evaporated to dryness and redissolved in 20% acetonitrile/water. The procedure used budesonide-d8 as internal standard and gave a 3.5-fold concentration of plasma to extract. The final extracts (5 μL injected) were analyzed with selected reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionization in positive mode. The chromatography system used a 100 mm ACQUITY BEH UPLC column and a gradient system consisting of aqueous 0.1% formic acid and acetonitrile as organic modifier. Phospholipid removal was found to be needed during method development in order to reduce ion suppression effects from matrix and to increase method sensitivity. The measuring range was 50-5000 pg/mL with and LOD 24 pg/mL. Calibration response showed good linearity (correlation coefficients<0.99) over the measuring range. The absolute recovery over the sample preparation procedure was estimated to 67%. Total imprecision was <9% at three levels and accuracy was between 98.9 and 103%. The method was successfully applied for analysis of 864 study samples in a short time. The quality control samples at concentration levels 200 and 2000 pg/mL gave a total imprecision of 7.4% and 4.2%, respectively, (n=95).

Laser Electrospray Mass Spectrometry Minimizes Ion Suppression Facilitating Quantitative Mass Spectral Response for Multicomponent Mixtures of Proteins

A comparison of the mass spectral response for myoglobin, cytochrome c, and lysozyme is presented for laser electrospray mass spectrometry (LEMS) and electrospray ionization-mass spectrometry (ESI-MS). Analysis of multicomponent protein solutions using nonresonant femtosecond (fs) laser vaporization with electrospray postionization mass spectrometry exhibited significantly reduced ion suppression effects in comparison with conventional ESI analysis, enabling quantitative measurements over 4 orders of magnitude in concentration. No significant charge reduction was observed in the LEMS experiment while the ESI measurement revealed charge reduction for myoglobin and cytochrome c as a function of increasing protein concentration. Conventional ESI-MS of each analyte from a multicomponent solution reveals that the ion signal detected for myoglobin and cytochrome c reaches a plateau and then begins to decrease with increasing protein concentration preventing quantitative analysis. The ESI mass spectral response for lysozyme from the mixture initially decreased, before increasing, with increasing multicomponent solution concentration.