In the analysis of proteome changes arising during the early stages of a biological process (e.g. disease or drug treatment) or from the indirect influence of an important factor, the biological variations of interest are often small (ϳ10%). The corresponding requirements for the precision of proteomics analysis are high, and this often poses a challenge, especially when employing label-free quantification. One of the main contributors to the inaccuracy of label-free proteomics experiments is the variability of the instrumental response during LC-MS/MS runs. Such variability might include fluctuations in the electrospray current, transmission efficiency from the air-vacuum interface to the detector, and detection sensitivity. We have developed an in silico post-processing method of reducing these variations, and have thus significantly improved the precision of label-free proteomics analysis. For abundant blood plasma proteins, a coefficient of variation of approximately 1% was achieved, which allowed for sex differentiation in pooled samples and Ϸ90% accurate dif- Label-free proteomics is sensitive, comprehensive, and versatile (1, 2). The "lyse, digest, and analyze" approach requires the least sample preparation and wet chemistry of all quantitative proteomics approaches (3). The label-free quantification technique is equally applicable to the analysis of peptide mixtures, protein complexes, body fluid proteomes, whole organisms, organs, and organelles. It also does not impose a limitation on the number of samples, which makes it best suited for the requirements of clinical proteomics in which analyses of large cohorts are common. Additionally, a single LC-MS/MS run can identify and quantify several thousand proteins, which makes the cost of analysis per quantified peptide, protein, or proteome very low (4).A significant drawback of label-free proteomics has been its limited precision in the determination of relative changes in peptide abundance, even when the area of the extracted chromatographic peak is used as the peptide abundance. It has been estimated that such label-free quantification gives abundance ratio results that are on average two to three times less accurate than the "gold standard" in global proteomics, stable isotope labeling of amino acids in cell culture (SILAC) 1 (4). There are several reasons for such a performance gap. Unlike in SILAC, where proteins in all samples under comparison are extracted and digested simultaneously (5, 6), in the label-free method, each sample is prepared independently, and therefore variations in sample preparation conditions can cause abundance fluctuations. However, these variations are a subjective factor that can be reduced by training the personnel and/or by employing sample preparation robots. The main objective contributor to the imprecision of a label-free LC-MS/MS experiment is the fluctuation of the instrumental response during the LC-MS/MS run or series of runs. A major component of the instrumental response fluctuation is the variation in the current...
Nonmuscle invasive tumors of the bladder often recur and thereby bladder cancer patients need regular re-examinations which are invasive, unpleasant, and expensive. A noninvasive and less expensive method, e.g. a urine dipstick test, for monitoring recurrence would thus be advantageous. In this study, the complementary techniques mass spectrometry (MS) and Western blotting (WB)/dot blot (DB) were used to screen the urine samples from bladder cancer patients. High resolving MS was used to analyze and quantify the urinary proteome and 29 proteins had a significantly higher abundance (p<0.05) in bladder cancer samples compared with control urine samples. The increased abundance found in urine from bladder cancer patients compared with controls was confirmed with Western blot for four selected proteins; fibrinogen β chain precursor, apolipoprotein E, α-1-antitrypsin, and leucine-rich α-2-glycoprotein 1. Dot blot analysis of an independent urine sample set pointed out fibrinogen β chain and α-1-antitrypsin as most interesting biomarkers having sensitivity and specificity values in the range of 66-85%. Exploring the Human Protein Atlas (HPA) also revealed that bladder cancer tumors are the likely source of these proteins. They have the potential of being useful in diagnosis, monitoring of recurrence and thus may improve the treatment of bladder tumors, especially nonmuscle invasive tumors.
Blood-based anti-amyloid-β (Aβ) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimer's disease (AD). IgG functionality can drastically change from anti- to pro-inflammatory via alterations in the IgG-Fc N-glycan structure. Herein, we tested if IgG-Fc glycosylation in plasma is indeed altered during the development of AD. Samples from age-matched subjects of 23 controls, 58 patients with stable mild cognitive impairment (SMCI), 34 patients with progressive (P)MCI, and 31 patients with AD were investigated. Label-free shotgun proteomics was applied without glycoprotein enrichment. Glycans on peptides EEQYNSTYR (IgG1) and EEQFNSTFR (IgG2) were quantified, and their abundances were normalized to total IgGn glycoform abundance. Univariate and multivariate statistics were employed to investigate the correlations between the patients groups and the abundances of the IgG glycoforms as well as those of inflammatory mediating proteins. Significant differences (p ≤ 0.05) were found, with a lower abundance of complex galactosylated and sialylated forms in AD. For females, a decline in glycoform complexity correlated with disease progress but an inverse change was found in males prior to the onset of AD. Principal component analysis (PCA; Males: R(2)X(cum) = 0.65, Q(2)(cum) = 0.34; Females: R(2)X(cum) = 0.62, Q(2)(cum) = 0.36), confirmed the gender similarities (for controls, SMCI and AD) as well as differences (for PMCI), and showed a close correlation between pro-inflammatory protein markers, AD, female PMCI, and truncated IgG-Fc glycans. The differences observed between genders prior to the onset of AD may indicate a lower ability in females to suppress peripheral inflammation, which may lead to exacerbated disease progression.
Increased levels of isoaspartyl residues (isoAsp) have previously been found in proteins of Alzheimer's disease (AD) brains and in blood proteins of patients suffering from uremia, the disease sharing common pathological features with AD. One can hypothesize that higher levels of isoAsp should be present in blood proteins of AD patients. Also, because of higher AD prevalence in females, they can be expected to have higher level of isoAsp than males. Here we modified our recently developed proteome-wide isoAsp analysis approach for testing these hypotheses. Eight blood plasma samples pooled from 218 individuals suffering from either mild cognitive impairment (MCI) or AD were analyzed by tandem mass spectrometry using electron transfer dissociation. Based on specific fragmentation pattern of isoAsp, the healthy controls were found to contain lower level of isoAsp compared with age-matched MCI and AD patients (p = 0.03). This result was further validated (p = 0.05) by 96 individual sample analyses, giving the combined value of p ≈ 0.01. Female pooled samples were found to contain higher level of isoAsp than male in both pooled and individual samples, with overall p ≈ 0.01. These findings verify the above hypotheses, and provide protein candidates for further investigation of the link between isoAsp and AD.
Significant sex bias in AD-specific biomarkers underscores the necessity of selecting sex-balanced cohort in AD biomarker studies, or using sex-specific models. Blood protein biomarkers are found to be promising for predicting AD progression in clinical settings.
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