Aberrant proteins in the saliva of patients with oral squamous cell carcinoma

Confirmation of oral squamous cell cancer (OSCC) currently relies on histological analysis, which does not provide clear indication of cancer development from precancerous lesions. In the present study, whole saliva proteins of patients with OSCC (n = 12) and healthy subjects (n = 12) were separated by 2DE to identify potential candidate biomarkers that are much needed to improve detection of the cancer. The OSCC patients’ 2DE saliva protein profiles appeared unique and different from those obtained from the healthy subjects. The patients’ saliva α1‐antitrypsin (AAT) and haptoglobin (HAP) β chains were resolved into polypeptide spots with increased microheterogeneity, although these were not apparent in their sera. Their 2DE protein profiles also showed presence of hemopexin and α‐1B glycoprotein, which were not detected in the profiles of the control saliva. When subjected to densitometry analysis, significant altered levels of AAT, complement C3, transferrin, transthyretin, and β chains of fibrinogen and HAP were detected. The increased levels of saliva AAT, HAP, complement C3, hemopexin, and transthyretin in the OSCC patients were validated by ELISA. The strong association of AAT and HAP with OSCC was further supported by immunohistochemical staining of cancer tissues. The differently expressed saliva proteins may be useful complementary biomarkers for the early detection and/or monitoring of OSCC, although this requires validation in clinically representative populations.     

Detection of mosaicism for the polymorphic variants in the 5′‐UTR of hOGG1 by cloning and sequence analysis and pyrosequencing

Mosaicism refers to the presence of genetically distinct cell lines within an organism or a tissue. Somatic mosaicism exists in distinct populations of somatic cells and commonly arises as a result of somatic mutations, mainly in early embryonic development. SNPs are important markers that distinguish between different individuals in heterogeneous biological samples and contribute greatly to disease risk association studies. In this work, we investigated the relationship between the functional variants in the 5′‐UTR of the hOGG1 gene and the risk of type 2 diabetes. Upon detection of the polymorphisms c.‐53G>C, c.‐23A>G, and c.‐18G>T in the hOGG1 gene, we found that mosaicism was present in 3/28 (10.71%), 7/51 (13.73%), and 1/44 (2.27%) patients respectively, who were carriers of these single nucleotide variations, by cloning and sequence analysis and pyrosequencing. Statistical analysis showed that the frequency of the variation c.‐23A>G in the hOGG1 5′‐UTR in type 2 diabetic patients was significantly higher than that in healthy controls. However, sequencing of the mutant alleles in mosaic individuals showed weak peaks that may affect detection of the SNPs and impair association‐based investigations.     

A further investigation on a MALDI‐based method for evaluation of markers of renal damage

The validity of the urinary protein profile to characterize the pathological states of diabetic, nephropathic and diabetic–nephropathic patients was considered on the basis of previously obtained results by MALDI/MS, showing a different abundance ratio of the collagen α1 and α5 chain precursor fragments at m/z 1219 and 2049 and of the uromodulin precursor fragment at m/z 1912 observed in healthy subjects and patients; a larger number of subjects was examined and the obtained results were statistically evaluated. The p values related to the observed differences indicate that they are statistically significant when comparing all patients versus healthy controls, diabetic with normo or microalbuminuria versus nephropathic with advanced renal disease patients and diabetic with normo or microalbuminuria versus diabetic with advanced nephropathy patients. The scatter plot matrix gives evidence of the strict inverse relationship between the abundances of ions at m/z 1912 and 1219, the correlation coefficient being particularly high (r = 0.921, p < 0.001). The relationship between the true positive rate (sensitivity) and false positive rate (1—specificity) for every possible cutoff value in abundance of the considered ionic species was investigated through the receiver‐operating characteristic (ROC) curve. The obtained data indicate that a good differentiation of nephropathic patients with advanced renal disease and diabetic patients with advanced nephropathy versus healthy subjects can be easily obtained by this approach. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a method for urine bikunin/urinary trypsin inhibitor (UTI) quantitation and structural characterization: Application to type 1 and type 2 diabetes

Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half‐life of few minutes and it is rapidly excreted into urine as urinary trypsin inhibitor (UTI). UTI levels are usually low in healthy individuals but they can increase up to tenfold in both acute and chronic inflammatory diseases. This article describes a sensitive method for both direct UTI quantitation and structural characterization. UTI purification was performed by anion exchange micro‐chromatography followed by SDS‐PAGE. A calibration curve for protein quantitation was set up by using a purified UTI fraction. UTI identification and structural characterization was performed by Nano‐LC‐MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetes, and 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients with respect to controls (p < 0.001 and p = 0.001, respectively). Spearman's correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method allows UTI quantitation from very low quantities of specimen. Furthermore, as UTI concentration is normalized for creatinine level, the analysis could be also performed on randomly collected urine samples. Finally, MS/MS analysis prospects the possibility of characterizing PTM sites potentially able to affect UTI localization, function, and pathophysiological activity. Preliminary results suggest that UTI levels could represent a useful marker of chronic inflammatory condition in type 1 and 2 diabetes.     

Proteomic identification of salivary transferrin as a biomarker for early detection of oral cancer

Oral cancer has a low five-year survival rate. Early detection of oral cancer could reduce the mortality and morbidity associated with this disease. Saliva, which can be sampled non-invasively and is less complex than blood, is a good potential source of oral cancer biomarkers. Proteomic analysis of saliva from oral cancer patients and control subjects was performed to identify salivary biomarkers of early stage oral cancer in humans. The protein profile of pooled salivary samples from patients with oral squamous cell carcinoma (OSCC) or OSCC-free control subjects was analyzed using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Potential biomarkers were verified by Western blotting and ELISA assays. Transferrin levels were elevated in the saliva of OSCC patients as determined using 2DE followed by MALDI-TOF MS and confirmed by MALDI-TOF/TOF MS, Western blotting and ELISA. The increase in salivary transferrin levels in OSCC patients strongly correlated with the size and stage of the tumor. The area under the receiver-operating characteristics curves showed that salivary transferrin-based ELISA was highly specific, sensitive and accurate for the early detection of oral cancer. We have identified salivary transferrin as a biomarker for the detection of early stage oral cancer. This finding provides a promising basis for the development of a non-invasive diagnostic test for early stage oral cancer.     

Detection of picomolar levels of interleukin-8 in human saliva by SPR

Researchers at UCLA have discovered that the levels of interleukin-8 (IL-8) protein in the saliva of healthy individuals and patients with oropharyngeal squamous cell carcinoma (OSCC) are 30 pM and 86 pM, respectively. In this study, we present the development of the first immunoassay for the quantification of picomolar IL-8 concentrations in human saliva using Biacore surface plasmon resonance (SPR) in a microfluidic channel. A sandwich assay using two monoclonal antibodies, which recognize different epitopes on the antigen (IL-8), was used. Only 13 minutes were required to determine the quantity of pure IL-8 added to just 100 microL of either buffer or saliva-based samples. The limit of detection (LOD) of this immunoassay in buffer was 2.5 pM, and the precision of the response for each concentration was <3% of the coefficient of variation. When first analyzing the saliva supernatants, non-specific binding to the surface was observed. By adding carboxymethyl dextran sodium salt (10 mg mL(-1)) to compete with the surface dextran and primary antibody for non-specific interactions, the signal to noise ratio was greatly improved. The LOD of this immunoassay in saliva was 184 pM. A minimum concentration of 250 pM of exogenous IL-8 could then be consistently detected in a salivary environment. The precision of the response for each IL-8 concentration tested was <7% of the coefficient of variation. Diagnostic sensitivity for oral cancer can be achieved by pre-concentrating the saliva samples 10 fold prior to SPR analysis, making the target levels of IL-8 300 pM for healthy individuals and 860 pM for oral cancer patients.     

The role of salivary peptides in dental caries

Dental caries is a complex disease, characterized by demineralization of tooth structure. With a protective role, several salivary phosphopeptides appear to be involved in remineralization processes, delaying the loss of tooth structure. In this work we have correlated peptide saliva composition with dental caries susceptibility through the analysis of saliva and hydroxyapatite-adsorbed salivary peptides samples. Saliva samples were obtained from two groups, a caries-free and a cariessusceptible group, and were analysed using HPLC-MS and a sequential extraction with 6 m of guanidine followed by tri fluoroacetate. Data analysis has allowed us to verify a strong correlation between large amounts phosphopeptides (PRP1/3, histatin 1 and statherin), and the absence of dental caries, which reinforces the importance of these peptides in the maintenance of tooth integrity. In addition, in the caries-susceptible group a high number of peptide fragments was observed, suggesting a high proteolytic activity.     

Verification of protein biomarker specificity for the identification of biological stains by quadrupole time‐of‐flight mass spectrometry

Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of “candidate biomarkers” specific to each of five body fluids (i.e ., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time‐of‐flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single‐source samples of these human body fluids were accurately identified by the detection of one or more high‐specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2‐component mixtures of human body fluids, the multiplex assay accurately identified both components in a single‐pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins.     

GC–MS Based Plasma Metabolic Profiling of Type 2 Diabetes Mellitus

Gas chromatography–mass spectrometry based metabolic profiling was used to characterize metabolic profiles in type 2 diabetes mellitus. We found distinct differences between type 2 diabetic patients and healthy controls, type 2 diabetic patients before and after treatment of the plasma metabolic profiles. Furthermore, levels of lactate, α-hydroxyisobutyric acid, phosphate, 1-monopalmitin and 1-monostearin were closely correlated with the disease state and may be considered as potential biomarkers for type 2 diabetes mellitus. The results demonstrated that plasma GC–MS metabolic profiling combined with multi- and uni-variate statistical analysis techniques provided an efficient way of depicting metabolic perturbations of diseases, and it may also be able to assist disease and treatment surveillance.     

The determination of salivary oxypurines before and after exercise by combined liquid chromatography-field asymmetric waveform ion mobility spectrometry-time-of-flight mass spectrometry

A method combining field asymmetric waveform ion mobility spectrometry with liquid chromatography-mass spectrometry (LC-FAIMS-MS) has been developed for the analysis of the oxypurine compounds hypoxanthine (HX) and xanthine (XA) in saliva. Separation of the oxypurines from interfering matrix components was investigated using FAIMS-MS. The selected FAIMS parameters were then applied to the rapid LC-FAIMS-MS analysis of HX and XA using a short chromatographic separation method (7 min). A comparison of the LC-MS method with and without FAIMS applied, resulted in improved discrimination from saliva matrix interferences and improved chromatographic peak integration for both HX and XA using a FAIMS separation. A quantitative evaluation of the LC-FAIMS-MS method was performed giving limits of detection of 2.0 ng mL^?1 for HX and 1.8 ng mL^?1 for XA, and limits of quantification of 6.6 ng mL^?1 for HX and 6.0 ng mL^?1 for XA. The developed LC-FAIMS-MS method was applied to the targeted analysis of the oxypurine metabolites in saliva collected from healthy male athletes (n =?11) before and after exercise designed to induce oxidative stress; post-exercise collection time-points included immediately after exercise, one hour and twenty-four hours’ post-exercise. The salivary concentrations of both HX and XA were lower after physical exercise, compared to the pre-exercise (rest) concentrations and returned to approximately pre-exercise levels after twenty-four hours. The method reported has the potential for monitoring the salivary oxypurines, HX and XA, as biomarkers of oxidative stress and in other clinical applications.     

Impact of Matrix Metalloproteinase-9 during Periodontitis and Cardiovascular Diseases

Matrix metalloproteinase-9 (MMP-9) has been shown to play a key role in endothelial function and perhaps pivotal in the correlation between periodontal disease and cardiovascular disease (CVD). For the study, the impact of MMP-9 of periodontitis and CVD on serum and saliva concentrations was analyzed. For the study patients with periodontitis (n = 31), CVD (n = 31), periodontitis + CVD (n = 31), and healthy patients (n = 31) were enrolled. Clinical and demographic characteristics as well as serum and salivary MMP-9 were evaluated. MMP-9 concentrations in serum and saliva were statistically elevated in patients with CVD (p < 0.01) and in patients with periodontitis plus CVD (p < 0.001) compared to patients with periodontitis and healthy subjects. Multivariate regression analysis showed that c-reactive protein (hs-CRP) was the only significant predictor for MMP-9 serum (p < 0.001), whereas hs-CRP (p < 0.001) and total cholesterol (p = 0.029) were the statistically significant salivary MMP-9 predictors. This study evidenced that patients with CVD and periodontitis + CVD presented elevated MMP-9 concentrations in serum and saliva compared to patients with periodontitis and healthy subjects. Furthermore, hs-CRP was a negative predictor of serum and salivary MMP-9.     

Electrochemical Biosensor Based on Bienzyme and Carbon Nanotubes Incorporated into an Os‐complex Thin Film for Continuous Glucose Detection in Human Saliva

Saliva opens a door for noninvasive and painless glucose testing since it reflects changes in the body physiology of diabetic individuals as compared to healthy ones. In this paper, a unique, disposable saliva biosensor has been developed for accurate, low cost, and continuous glucose monitoring. The biosensor exhibits linear dependence of the catalytic current upon glucose bulk concentration over the 0.05–1.5 mM range (R=0.998). A detection limit of 0.003 mM can be calculated considering three times the standard deviation of the blank signal divided by the sensitivity of the sensor. The selectivity of the biosensor was evaluated by adding the interferent species of lactate, ascorbic acid and uric acid into in 0.5 mM glucose; the nearly negligible interference current indicates its good selectivity. The operational stability of the biosensor was measured in 1 mM glucose over a 2 h period (RSD=3.27 %). A clinical trial on real‐time noninvasive salivary glucose monitoring was carried out on 30 individuals by measuring subjects’ salivary glucose and blood glucose in parallel. The results show that there is a good correlation of glucose levels in saliva and in blood 2 h after breakfast. Thus, the disposable biosensor would be a potential alternative for continuous glucose detection in human saliva.     

Serum metabolomics of laryngeal cancer based on liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry

The discovery of new laryngeal cancer‐related metabolite biomarkers could help to facilitate early diagnosis. A serum metabolomics study from laryngeal cancer patients and healthy individuals was conducted using liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry. Univariate and multivariate statistics were used to discriminate laryngeal cancer patients and healthy individuals. 1‐Palmitoyl‐sn‐glycero‐3‐phosphocholine (LysoPC 16:0), 1‐o‐hexadecyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (PAF) and 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine were found to be significantly different between the laryngeal cancer group and the healthy group. They are mainly involved in phospholipids catabolism, linoleic acid metabolism, α‐linoleic acid metabolism and arachidonic acid metabolism. The area under the curve of the biomarker combined by two metabolites (LysoPC 16:0 and PAF) was 0.935, the sensitivity was 0.962 and the specificity was 0.825. LysoPC 16:0 and PAF may show diagnostic potential for laryngeal carcinoma.     

Acute‐phase proteins investigation based on lectins affinity capture prior to 2‐DE separation: Application to serum from multiple sclerosis patients

Plasma acute‐phase proteins (APPs) glyco‐isoforms are important biomarkers of inflammatory processes such as those occurring in multiple sclerosis (MS). Specific analysis of these proteins is often hampered by sample biochemical complexity. The aim of our study was to set up a method to accurately visualize, identify and quantify APPs glyco‐isoforms in human serum. An enrichment strategy based on affinity chromatography using the carbohydrate‐binding proteins concanavalin A (ConA) and erythrina cristagalli lectin (ECL) was applied to pooled serum samples from 15 patients and 9 healthy individuals. Image analysis of 2‐DE detected 30 spots with a fold change higher than 1.5. A total of 14 were statistically significant (p value<0.05): 7 up‐regulated and 7 down‐regulated in MS samples. ESI LC‐Nanospray IT mass spectrometry analysis confirmed that all of them were APPs isoforms supporting the idea that the accurate analysis of differential glycosylation profiles in these biomarkers is instrumental to distinguish between MS patients and healthy subjects. Additionally, overlaps in ConA/ECL maps protein patterns suggest how the used lectins are able to bind sugars harbored by the same oligosaccharide structure. Among identified proteins, the presence of complex and/or hybrid type N‐linked sugar structures is well known. Performing galectin‐3 binding and Western blotting, we were able to demonstrate a correlation between hybrid type glyco‐isoforms of β‐haptoglobin and MS. In conclusion, although the patho‐physiological role of the identified species still remains unclear and further validations are needed, these findings may have a relevant impact on disease‐specific marker identification approaches.     

Low molecular weight proteins in urines from healthy subjects as well as diabetic,nephropathic and diabetic‐nephropathic patients: a MALDI study

Urine samples from healthy subjects as well as diabetic, nephropathic and diabetic‐nephropathic patients were analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry in order to establish evidence of some possible differences in the peptide profile related to the pathological states. Multivariate analysis suggested the possibility of a distinction among the considered groups of patients. Some differences have been found, in particular, in the relative abundances of three ions at m/z 1912, 1219 and 2049. For these reasons, further investigation was carried out by MALDI/TOF/TOF to determine the sequence of these peptides and, consequently, to individuate their possible origin. By this approach, the peptide at m/z 1912 was found to originate from uromodulin, and its lower expression in the case of nephropathy can be well related to the pathological condition. Ions at m/z 2049 and 1219 originate from the collagen α‐1(I) chain precursor and from the collagen α‐5 (IV) chain precursor, respectively, and, also in this case, their different expressions can be related to the pathologies under investigation. The obtained data seem to indicate that urine is an interesting biological fluid to investigate on the peptide profile and to obtain, consequently, information on the dismetabolism activated by specific pathologies. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,characterization, antioxidant and anti‐diabetic activities of a novel protein–vanadium complex

The protein–vanadium complex was successfully synthesized and systematically characterized using electron paramagnetic resonance, Fourier transform‐infrared spectroscopy and thermogravimetric analysis. The antioxidant activity analysis indicated that it had better radical scavenging activity on 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH), 2,2′‐azinobis (3‐ethylbenzothiazoline)‐6‐sulfonic acid (ABTS) and O₂^? compared with the free protein and vanadate. Additionally, the complex exhibited high anti‐diabetic activity against Saccharomyces cerevisiae α‐glucosidase and rat intestinal maltase with IC₅₀ values of 258.53 and 72.41 μg/ml, respectively. Kinetics study showed that the complex was a mixed inhibition type against S. cerevisiae α‐glucosidase and uncompetitive inhibition type against rat intestinal maltase. These indicated that the complex with antioxidant and anti‐diabetic potential could be used for lowering blood glucose that might be caused by insufficient secretion of insulin in the body or excess fat storage. Our findings provide a new protein–vanadium complex for further use in diabetes mellitus or obesity.     

Two-dimensional analysis of tear protein patterns of diabetic patients.

In diabetic patients, dry eye and other ocular surface diseases occur more often than in healthy subjects. The aim of this study was to analyze the tear protein patterns of patients suffering from diabetes mellitus type II (dia) and to compare them to the patterns of healthy volunteers (ctrl). Tear proteins of nonstimulated tears of 20 patients (ctrl n=10, dia n=10) were separated using two-dimensional electrophoretic techniques. The protein patterns of each group were analyzed by a multivariate analysis of discriminance. Furthermore, for all spots of each gel, a 50 x 50 variables pH/Mr (molecular weight) array was generated and subsequently analyzed by a multivariate analysis of discriminance. Additionally, an artificial neural network was trained using the matrix data as input and a sensitivity analysis was performed to figure out, which spots were the most important to differentiate between the tear protein patterns. In both groups a complex staining pattern could be obtained. In diabetic patients significantly more spots were detected compared to the control group (P<0.02). The analysis of discriminance found a highly significant difference between dia and ctrl (P<0.00001). Using the matrix data, the analysis of discriminance showed a significant difference between the two groups, too (P<0.0001). The sensitivity analysis by means of the artificial neural network revealed several spots that were more expressed or more frequently present in the diabetic group. Our findings reveal that the composition of tear proteins of diabetic patients is different from that of healthy subjects. The use of the two-dimensional electrophoretic technique could give more insight into the diabetic-related changes in the tear film composition.     

Investigation of bacterial viability from incubated saliva by application of flow cytometry and hyphenated separation techniques

The aim of the study was determination of bacterial viability in saliva samples and finding a correlation between microbiological and volatile profiles of saliva depending on incubation time. Bacteria colonizing healthy oral cavities were also identified. Twelve healthy adults donated unstimulated saliva samples. Flow cytometry, optical density measurements and colony‐forming unit (CFU) counting method were employed for analyses of native and inoculated saliva after 0, 1, 2, 24, and 48 h of incubation. Volatile profiles were acquired using headspace‐solid phase microextraction‐gas chromatography/mass spectrometry (HS‐SPME‐GC/MS). Oral bacteria were the most viable within 2 h after collection of saliva. Extension of incubation time to 48 h caused considerable decrease in live bacteria counts and sharp increase in dead bacteria counts. The most prevalent strain was Sphingomonas paucimobilis (26.67%). The number of volatiles raised from 5 to 27 with incubation time and most of them were putrefaction products, such as methanethiol, indole and pyrrole. HS‐SPME‐GC/MS method is insufficient for volatile profiling of “fresh” saliva and should be directed rather to investigation of bacterial metabolites.     

Study of human neutrophil peptides in saliva by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides – HNP 1, 2, and 3 – in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI‐TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva‐absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age‐dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging. Copyright © 2009 John Wiley & Sons, Ltd.     

Phospholipidomic identification of potential plasma biomarkers associated with type 2 diabetes mellitus and diabetic nephropathy

Type 2 diabetes mellitus (T2DM) and its attendant complications, such as diabetic nephropathy (DN), impose a significant societal and economic burden. The investigation of discovering potential biomarkers for T2DM and DN will facilitate the prediction and prevention of diabetes. Phospholipids (PLs) and their metabolisms are closely allied to nosogenesis and aggravation of T2DM and DN. The aim of this study is to characterize the human plasma phospholipids in T2DM and DN to identify potential biomarkers of T2DM and DN. Normal phase liquid chromatography coupled with time of flight mass spectrometry (NPLC-TOF/MS) was applied to the plasma phospholipids metabolic profiling of T2DM and DN. The plasma samples from control (n = 30), T2DM subjects (n = 30), and DN subjects (n = 52) were collected and analyzed. The significant difference in metabolic profiling was observed between healthy control group and DM group as well as between control group and DN group by the help of partial least squares discriminant analysis (PLS-DA). PLS-DA and one-way analysis of variance (ANOVA) were successfully used to screen out potential biomarkers from complex mass spectrometry data. The identification of molecular components of potential biomarkers was performed on Ion trap-MS/MS. An external standard method was applied to quantitative analysis of potential biomarkers. As a result, 18 compounds in 7 PL classes with significant regulation in patients compared with healthy controls were regarded as potential biomarkers for T2DM or DN. Among them, 3 DM-specific biomarkers, 8 DN-specific biomarkers and 7 common biomarkers to DM and DN were identified. Ultimately, 2 novel biomarkers, i.e., PI C18:0/22:6 and SM dC18:0/20:2, can be used to discriminate healthy individuals, T2DM cases and DN cases from each other group.