Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100?000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14?000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.     

The Application of Gaussian Mixture Models for Signal Quantification in MALDI-ToF Mass Spectrometry of Peptides

Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) coupled with stable isotope standards (SIS) has been used to quantify native peptides. This peptide quantification by MALDI-TOF approach has difficulties quantifying samples containing peptides with ion currents in overlapping spectra. In these overlapping spectra the currents sum together, which modify the peak heights and make normal SIS estimation problematic. An approach using Gaussian mixtures based on known physical constants to model the isotopic cluster of a known compound is proposed here. The characteristics of this approach are examined for single and overlapping compounds. The approach is compared to two commonly used SIS quantification methods for single compound, namely Peak Intensity method and Riemann sum area under the curve (AUC) method. For studying the characteristics of the Gaussian mixture method, Angiotensin II, Angiotensin-2-10, and Angiotenisn-1-9 and their associated SIS peptides were used. The findings suggest, Gaussian mixture method has similar characteristics as the two methods compared for estimating the quantity of isolated isotopic clusters for single compounds. All three methods were tested using MALDI-TOF mass spectra collected for peptides of the renin-angiotensin system. The Gaussian mixture method accurately estimated the native to labeled ratio of several isolated angiotensin peptides (5.2% error in ratio estimation) with similar estimation errors to those calculated using peak intensity and Riemann sum AUC methods (5.9% and 7.7%, respectively). For overlapping angiotensin peptides, (where the other two methods are not applicable) the estimation error of the Gaussian mixture was 6.8%, which is within the acceptable range. In summary, for single compounds the Gaussian mixture method is equivalent or marginally superior compared to the existing methods of peptide quantification and is capable of quantifying overlapping (convolved) peptides within the acceptable margin of error.     

Quantitative analysis of backbone-cyclised peptides in plants

Progress in understanding the biosynthetic pathway of the cyclotides has been hampered as this unique family of cyclic plant peptides are notoriously difficult to analyse by standard proteomic approaches such as gel electrophoresis. We have developed a simple, rapid and robust strategy for the quantification of cyclotides in crude plant extracts using MALDI-TOF MS making use of generic peptides similar in mass to the analyte as internal standards for calibration. Linearity (r(2)>0.99) over two orders of magnitude (down to femtomole levels) was achieved in plant extracts, allowing quantitative analysis of transgenic and endogenous peptide expression.     

Affinity-tagged phosphorylation assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ATPA-MALDI): application to calcium/calmodulin-dependent protein kinase

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based kinase assay using a peptide substrate tagged with a biotinyl group has been developed. The peptide moiety was designed to serve as an efficient substrate for calcium/calmodulin-dependent protein kinase II, based on the in vivo phosphorylation site of phosrestin I, a Drosophila homolog of arrestin. In the assay, the quantitative relationship was determined from the ratio of the peak areas between the two peaks respectively representing the unphosphorylated and the phosphorylated substrate. Attempts to assay phosphorylated peptides directly from the reaction mixture, gave inaccurate results because of the high noise level caused by the presence of salts and detergents. In contrast, after purifying the substrate peptides with the biotin affinity tag using streptavidin-coated magnetic beads, peak areas accurately represented the ratio between the unphosphorylated and phosphorylated peptide. By changing the substrate peptide to a peptide sequence that serves as a kinase substrate, it is expected that an efficient non-radioactive protein kinase assay using MALDI-TOF MS can be developed for any type of protein kinase. We call this technique "Affinity-Tagged Phosphorylation Assay by MALDI-TOF MS (ATPA-MALDI)." ATPA-MALDI should serve as a quick and efficient non-radioactive protein kinase assay by MALDI-TOF MS.     

Evaluation of the possible proteomic application of trypsin from Streptomyces griseus

Trypsin (EC 3.4.21.4) is the protease of choice for proteome analysis using mass spectrometry of peptides in sample digests. In this work, trypsin from Streptomyces griseus (SGT) was purified to homogeneity from pronase. The enzyme was evaluated in in-gel digestion of protein standards followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analyses of the digests. We recognized a remarkable cleavage performance of SGT. The number of produced and matching tryptic peptides was higher than in the case of commonly used bovine trypsin (BT) and allowed us to obtain higher identification scores in database searches. Interestingly, SGT was found to also generate nonspecific peptides whose sequencing by MALDI-TOF/TOF tandem mass spectrometry (MS/MS) revealed a partial F-X, Y-X, and W-X cleavage specificity. To suppress autolysis, either arginine or arginine plus lysine residues in SGT were modified by chemical reagents. In consequence, the autolytic pattern of SGT was reduced significantly, but specific activity dropped dramatically. As demonstrated by relative quantification of peptides at different times, SGT is more stable at 37 °C than is its bovine counterpart. We conclude that SGT represents a convenient alternative for proteomic applications involving protein digestion. Moreover, parallel digestions of sample aliquots by SGT and BT provide the possibility of combining partially different results (unique matching peptides) to improve protein identification.     

Quantitative evaluation of sphingomyelin and glucosylceramide using matrix-assisted laser desorption ionization time-of-flight mass spectrometry with sphingosylphosphorylcholine as an internal standard. Practical application to tissues from patients with Niemann-Pick disease types A and C, and Gaucher disease

Niemann-Pick disease types A and C, and Gaucher disease are glycolipid storage disorders characterized by the systemic deposition of glycosphingolipids, i.e., sphingomyelin in Niemann-Pick disease types A and C tissues and glucosylceramide in Gaucher disease ones, respectively. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we analyzed the sphingolipids in liver and spleen specimens from patients with Niemann-Pick disease types A and C, and Gaucher disease. Crude lipids were extracted from tissue containing 5mg protein with chloroform and methanol. After mild alkaline treatment of the crude lipids, a sphingolipid fraction was prepared and analyzed by MALDI-TOF/MS. The results were as follows: (a) ion peaks with m/z values corresponding to different sphingomyelin and ceramide monohexoside (CMH) species were clearly detected. (b) With sphingosylphosphorylcholine as the internal standard for quantification of sphingomyelin and CMH, the relative peak heights of sphingomyelin and CMH were calculated and plotted versus their contents. The relative peak heights of sphingomyelin and CMH showed linearity between 50 and 1500 ng sphingomyelin content, and between 5 and 150 ng CMH content, respectively. (c) Quantitative analysis revealed the accumulation of sphingomyelin in the liver and spleen specimens from the patients with Niemann-Pick disease types A and C. Striking accumulation of CMH was also detected in the liver and spleen specimens from the patients with Gaucher disease. This investigation indicated that accumulated sphingomyelin and CMH in small amounts of tissues from sphingolipidosis patients can be detected quantatively with the MALDI-TOF/MS method. This method will be useful not only for the diagnosis but also for biochemical pathophysiology evaluation of patients with various sphingolipidosis.     

P99-T A Simple Method to Quantitate Selenomethionine Incorporation into Proteins

Selenomethionine substitution is the preferred method for preparing heavy-atom derivatives of proteins for crystal structure determination using the multi-wavelength anomalous diffraction phasing method. This approach allows researchers to take advantage of the anomalous signal from a number of diverse atoms. We recently published a protocol describing a number of variables that play a role in determining incorporation efficiency of selenomethionine into mammalian expression systems.¹ Here we describe, in detail, a simple method for assessing selenomethionine substitution by replacement of methionine in homogeneous protein preparations. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF) technology following trypsin proteolysis of the recombinant protein, we are able to evaluate variables that play roles in affecting selenomethionine incorporation. Examples will illustrate (a) the ease of identification of modified peptides containing the selenomethione and (b) relative quantitation of such peptides when compared with the control, unmodified peptides.     

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of G_i2 (a heterotrimeric G-protein α-subunit) is described in detail.     

A direct approach to quantification of the cellular uptake of cell-penetrating peptides using MALDI-TOF mass spectrometry

This protocol allows the accurate quantification of cell-penetrating peptide (CPP) cellular uptake by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Quantification is based on the use of an internal standard with same chemical structure as the analyte but labeled with a stable isotope. The analyte and the standard can both be obtained by standard solid-phase peptide synthesis using commercially available amino acids. They are functionalized by biotin to allow their easy purification before MALDI-TOF MS analysis. The method allows determination of the amount of intact internalized peptide and the identification of potential intracellular digests. It can be used to simultaneously compare the uptake of several peptides, and can also be applied to the quantification of peptidic cargoes and the study of their intracellular stability. It is therefore a potent tool to study the mechanisms of CPPs internalization and to select new carriers for drug delivery. This protocol will take approximately 5 hours for the analysis of 12 samples (not including the time for cell incubation with peptides).     

Quantification of thymosin β4 in human cerebrospinal fluid using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been applied to the analysis of a wide range of biomolecules. To date, there are two specific areas of application where MALDI-TOF-MS is viewed as impractical: analysis of low-mass analytes and relative quantitative applications. However, these limitations can be overcome and quantification can be routine. Increased levels of thymosin β₄ (TB4) have been recently found in cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients. Our objective was to apply a label-free quantitative application of MALDI-TOF-MS to measure TB4 levels in human CSF by adding the oxidized form of TB4 as an internal standard. The relative peak area or peak height ratios of the native TB4 to the added oxidized form were evaluated. Considering the relative peak area ratios, healthy individuals showed a mean value of 40.8 ± 21.27 ng/ml, whereas CJD patients showed high values with a mean of 154 ± 59.07 ng/ml, in agreement with the previous observation found in CJD patients. Similar results were obtained considering peak height ratios. The proposed method may provide a simple and rapid screening method for quantification on CSF of TB4 levels suitable for diagnostic purposes.     

基质辅助激光解吸电离飞行时间质谱技术用于常见益生菌鉴定的初步研究

目的建立基质辅助激光解吸电离飞行时间质谱(MADLI-TOF MS)技术鉴定常见益生菌的实验方法并对MADLI-TOF MS技术的适用性进行初步评价。方法对MADLI-TOF MS技术鉴定常见益生菌过程中各影响因素进行考察,筛选出最佳的实验条件。利用19株供试菌株所得的蛋白指纹图谱对MADLI-TOF MS技术的适用性进行研究。结果建立了MADLI-TOF MS技术鉴定常见益生菌的最佳实验方法。初步证明MADLI-TOF MS技术具备在属、种、亚种以及菌株水平上鉴定常见益生菌的能力。结论建立的实验方法稳定性高、重复性好,可以作为MADLI-TOF MS技术鉴定常见益生菌的参考方法。MADLI-TOF MS技术可以作为常见益生菌鉴定的方法之一。     

Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents

The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.     

IQcat: multiplexed protein quantification by isoelectric QconCAT

Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by pI to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (～2 weeks), inexpensive and scalable purification of arg/lys labeled IQcat standards in the Escherichia coli auxotroph AT713. With this pipeline, we assess the fidelity of IQcat-based absolute quantification for ten yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.     

Introducing AAA-MS, a rapid and sensitive method for amino acid analysis using isotope dilution and high-resolution mass spectrometry

Accurate quantification of pure peptides and proteins is essential for biotechnology, clinical chemistry, proteomics, and systems biology. The reference method to quantify peptides and proteins is amino acid analysis (AAA). This consists of an acidic hydrolysis followed by chromatographic separation and spectrophotometric detection of amino acids. Although widely used, this method displays some limitations, in particular the need for large amounts of starting material. Driven by the need to quantify isotope-dilution standards used for absolute quantitative proteomics, particularly stable isotope-labeled (SIL) peptides and PSAQ proteins, we developed a new AAA assay (AAA-MS). This method requires neither derivatization nor chromatographic separation of amino acids. It is based on rapid microwave-assisted acidic hydrolysis followed by high-resolution mass spectrometry analysis of amino acids. Quantification is performed by comparing MS signals from labeled amino acids (SIL peptide- and PSAQ-derived) with those of unlabeled amino acids originating from co-hydrolyzed NIST standard reference materials. For both SIL peptides and PSAQ standards, AAA-MS quantification results were consistent with classical AAA measurements. Compared to AAA assay, AAA-MS was much faster and was 100-fold more sensitive for peptide and protein quantification. Finally, thanks to the development of a labeled protein standard, we also extended AAA-MS analysis to the quantification of unlabeled proteins.     

Difference in mass analysis using labeled lysines (DIMAL-K): a new, efficient proteomic quantification method applied to the analysis of astrocytic secretomes

Here we describe an original strategy for unbiased quantification of protein expression called difference in mass analysis using labeled lysine (K) (DIMAL-K). DIMAL-K is based on the differential predigestion labeling of lysine residues in complex protein mixtures. The method is relevant for proteomic analysis by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Protein labeling on lysine residues uses two closely related chemical reagents, S-methyl thioacetimidate and S-methyl thiopropionimidate. Using protein standards, we demonstrated that 1) the chemical labeling was quantitative, specific, and rapid; 2) the differentially labeled proteins co-migrated on two-dimensional gels; and 3) the identification by mass fingerprinting and the relative quantification of the proteins were possible from a single MALDI-TOF mass spectrum. The power of the method was tested by comparing and quantifying the secretion of proteins in normal and proinflammatory astrocytic secretomes (20 microg). We showed that DIMAL-K was more sensitive and accurate than densitometric image analysis and allowed the detection and quantification of novel proteins.     

Mineral oil-, glycerol-, and Vaseline-coated plates as matrix-assisted laser desorption/ionization sample supports for high-throughput peptide analysis

A novel protocol for rapid and high-quality sample preparation prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed by coating bare stainless steel plates with one of three adhesives: mineral oil, glycerol, or Vaseline. The advantages of these three adhesive coats are that they take little time to both prepare and wipe away, hold the matrices to prevent them from flying from the support, reduce the background matrix, and affect neither the resolution of the peptide peaks nor the accuracy of their determined molecular masses. Consequently, the signal intensity, detection limit, and tolerance of the analytes to contaminants on the three adhesive-coated plates are improved. In the two strategies of on-plate desalting and concentration of the peptide mixture, all three adhesives reduced the loss of peptides, especially in the case of larger molecular mass peptides. The microscope and stereomicroscope images of the deposited droplets showed that after dropping onto the adhesive coats, the droplets formed a reduced spot size, were more homogeneous, and showed sticky crystallization. Therefore, this is an easy-to-use, reproducible, highly sensitive, tolerant (to salts), and high-throughput method of peptide sample preparation for MALDI-TOF MS analysis.     

Detection of protein modifications and counterfeit protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry: studies of insulins

Isotope tags for relative and absolute quantification (iTRAQ) reagent coupled with matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, and Lantus) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI-TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including human insulin (P28K29) and Lispro insulin (K28P29), which differ only by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.     

Assay of protein tyrosine phosphatases by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A nonradioactive assay for protein tyrosine phosphatases (PTPs), employing a tyrosine-phosphorylated peptide as a substrate, has been developed and applied to analyze purified enzymes, cell extracts, and immunoprecipitates. The reaction was followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a linear and positive ion mode with delayed extraction. MALDI-TOF MS detects a loss of peptide mass by 80 Da as a result of dephosphorylation and, more importantly, it yields phospho-peptide to dephosphorylated product peak intensity ratios proportional to their concentration ratios. A strong bias of the MALDI-TOF MS toward detection of the non-phospho-peptide allows accurate detection of small fractions of dephosphorylation. The method is highly sensitive and reproducible. It can be applied to general assays of protein phosphatases with various phospho-peptides as substrates.     

Qualitative and quantitative comparison of brand name and generic protein pharmaceuticals using isotope tags for relative and absolute quantification and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry

The capability of iTRAQ (isotope tags for relative and absolute quantification) reagents coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) as a qualitative and quantitative technique for the analysis of complicated protein pharmaceutical mixtures was evaluated. Mixtures of Somavert and Miacalcin with a small amount of bovine serum albumin (BSA) as an impurity were analyzed. Both Somavert and Miacalcin were qualitatively identified, and BSA was detected at levels as low as 0.8 mol%. Genotropin and Somavert were compared in a single experiment, and all of the distinct amino acid residues from the two proteins were readily identified. Four somatropin drug products (Genotropin, Norditropin, Jintropin, and Omnitrope) were compared using the iTRAQ/MALDI-MS method to determine the similarity between their primary structures and quantify the amount of protein in each product. All four product samples were well labeled and successfully compared when a filtration cleanup step preceded iTRAQ labeling. The quantitative accuracy of the iTRAQ method was evaluated. In all cases, the accuracy of experimentally determined protein ratios was higher than 90%, and the relative standard deviation (RSD) was less than 10%. The iTRAQ and global internal standard technology (GIST) methods were compared, and the iTRAQ method provided both higher sequence coverage and enhanced signal intensity.     

A relative and absolute quantification of neutral N-linked oligosaccharides using modification with carboxymethyl trimethylammonium hydrazide and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantification of oligosaccharides is of great importance to investigate variations or changes in the glycans of glycoconjugates. Mass spectrometry (MS) has been widely applied to identification and structural analysis of complex oligosaccharides. However, quantification using MS alone is still quite challenging due to heterogeneous charge states and different ionization efficiency of various types of oligosaccharides. To overcome such shortcomings, derivatization with carboxymethyl trimethylammonium hydrazide (Girard’s reagent T [GT]) was introduced to generate a permanent cationic charge at the reducing end of neutral oligosaccharides, resulting in mainly [M]⁺ ion using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), so that the ambiguities caused by metal adduct peaks such as [M+K]⁺ and [M + Na]⁺ were avoided. To verify our method, the relative and absolute quantification of neutral glycans from human immunoglobulin G (IgG) and ovalbumin with internal standards of dextran ladders using MALDI-TOF MS were compared with those performed by conventional normal-phase high-performance liquid chromatography (NP-HPLC) profiling. The quantification using GT derivatization and MALDI-TOF MS agreed well with the HPLC profiling data and showed excellent reliability and reproducibility with better resolution and sensitivity. This method was further applied to quantify the enzymatically desialylated N-glycans from miniature pig kidney membrane proteins. The results showed that the low-abundance structures that could not be resolved by NP-HPLC were quantified with high sensitivity. Thus, this novel method of using modification of neutral sugars with GT is quite powerful for neutral glycan analysis, especially to quantify minute glycan samples with undetectable levels using HPLC.