We report the shotgun proteomic analysis of mammalian cell lysates that contain low nanogram amounts of protein. Proteins were denatured using methanol, digested using immobilized trypsin, and analyzed by UPLC-ESI-MS/MS. The approach generated more peptides and higher sequence coverage for a mixture of three standard proteins than the use of free trypsin solution digestion of heat- or urea-denatured proteins. We prepared triplicate RAW 264.7 cell lysates that contained 6 ng, 30 ng, 120 ng, and 300 ng of protein. An average of 2 ± 1, 23 ± 2, 134 ± 11, and 218 ± 26 proteins were detected for each sample size, respectively. The numbers of both protein and peptide IDs scaled linearly with the amount of sample taken for analysis. Our approach also outperformed traditional methods (free trypsin digestion of heat- or urea-denatured proteins) for 6 ng to 300 ng RAW 264.7 cell protein analysis in terms of number of peptides and proteins identified. The use of accurate mass and time (AMT) tags resulted in the identification of an additional 16 proteins based on 20 peptides from the 6 ng cell lysate prepared with our approach. When AMT analysis was performed for the 6 ng cell lysate prepared with traditional methods, no reasonable peptide signal could be obtained. In all cases, roughly ~30% of the digested sample was taken for analysis, corresponding to the analysis of a 2 ng aliquot of homogenate from the 6 ng cell lysate.
We report the performance of capillary zone electrophoresis coupled with an electrokinetically pumped electrospray interface and an Orbitrap-Velos mass spectrometer for high sensitivity protein analysis. We first investigated the system for quantitation of the tryptic digest of bovine serum albumin (BSA). The system produced outstanding linearity with respect to peak height, number of peptide IDs, and spectral counts across the range of 12 nM to 750 nM (60 amol to 3.5 fmol) of BSA injected. One peptide produced a detection limit of 0.3 nM (1.5 amol) injected. We also analyzed 700 pg of a tryptic digest prepared from a RAW264.7 cell lysate; 10 proteins were identified in triplicate analyses after filtering the data with peptide confidence value as high. This sample size corresponds to the protein content of ~10 eukaryotic cells.
Electrokinetically driven sheath flow interface; CZE-ESI-MS/MS; Protein digests
We demonstrate the use of capillary zone electrophoresis with an electrokinetic sheath-flow electrospray interface coupled to a triple quadrupole mass spectrometer for the accurate and precise quantification of leu-enkaphalin in a complex mixture using multiple-reaction monitoring (MRM). Assay time is <6 minutes, with no re-equilibration required between runs. A standard curve of Leu-enkephalin was performed in the presence of a background tryptic digest of bovine albumin. We demonstrate reasonably reproducible peak heights (21% relative standard deviation), retention times (better than 1% relative standard deviation), and robust electrospray quality. Our limit-of detection (3σ) was 60 pM, which corresponds to the injection of 115 zeptomole of peptide. This is a 10–20-fold improvement in mass sensitivity than we have obtained by nano HPLC/MRM and substantially better than reported for LC/MS/MS. Further quantification was performed in the presence of stable-isotope labeled versions of the peptides; under these conditions, linearity was observed across nearly four orders of magnitude. The concentration detection limit was 240 pM for the stable-isotope labeled quantification.
Motivation: There is now a large literature on statistical methods for the meta-analysis of genomic data from multiple studies. However, a crucial assumption for performing many of these analyses is that the data exhibit small between-study variation or that this heterogeneity can be sufficiently modelled probabilistically.
Results: In this article, we propose ‘assumption weighting’, which exploits a weighted hypothesis testing framework proposed by Genovese et al. to incorporate tests of between-study variation into the meta-analysis context. This methodology is fast and computationally simple to implement. Several weighting schemes are considered and compared using simulation studies. In addition, we illustrate application of the proposed methodology using data from several high-profile stem cell gene expression datasets.
We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reversed phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR<0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analysis each using 250 ng of the unfractionated peptide mixture at 95% confidence (FDR<0.15%). 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular mass. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for CE proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity.
Capillary electrophoresis can provide fast and efficient separations of peptides. However, the high speed separation and limited loading capacity of capillary electrophoresis requires the use of a fast and sensitive detector. While laser-induced fluorescence provides exquisite sensitivity and millisecond response time, it inherently generates a low information content signal. In contrast, mass spectrometry provides an information rich signal that is attractive for peptide analysis. The recently introduced Velos-Orbitrap mass spectrometer is capable of fast and sensitive tandem MS acquisition and simultaneous high accuracy MS acquisition, which is well suited for coupling with fast and efficient separation methods for peptide analysis. We evaluated this instrument as a detector for peptide separation by capillary electrophoresis. In MS mode, we observed low attomole detection limits for a number of peptides in a tryptic digest of standard proteins with high mass resolution (30,000 at m/z 400). The response time of the Orbitrap at this resolution was ~0.70 seconds, which was adequate to reconstruct the peak shape and area of our electrophoretic peaks. The linear ion-trap successfully recorded tandem MS spectra of tryptic peptides at 20 nM concentration.
Aldehyde- and NHS-activated magnetic microspheres were used to immobilize trypsin (CHO-trypsin and NHS-trypsin), and their performance for protein digestion was evaluated by reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry using an LTQ Orbitrap Velos instrument. NHS-trypsin provided greater sequence coverage and identified more peptides for the digestion of bovine serum albumin. A one-minute digestion at room temperature using the immobilized trypsin also identified more peptides (96 ± 6 vs. 48 ± 1) and produced higher sequence coverage (90 ± 2% vs. 75 ± 2%) than traditional free trypsin digestion for 12 hours at 37 °C. Analysis of 15 nM (0.001 mg/mL) BSA digested by NHS-trypsin in 1 min. at room temperature consistently yielded one detected peptide; 150 nM BSA generated 22 peptides. Peptide intensity and protein spectral count were used to evaluate the run-to-run digestion reproducibility of NHS-trypsin with a three-protein-mixture. Three high intensity peptides for each protein generated intensity ratios from 0.70 to 1.09 and spectral count ratios from 0.78 to 1.18. Finally, RAW 264.7 cell lysates were digested by NHS-trypsin for 10 min. and 30 min. at room temperature; 604 and 697 protein groups, respectively, were identified by RPLC-ESI-MS/MS, with a peptide false discovery rate of less than 1%. Digestion by solution phase trypsin for 12 hours at 37 °C resulted in identification of 878 protein groups.
protein digestion; trypsin-immobilized magnetic microspheres; reproducibility; RPLC-ESI-MS/MS
Conventional prenatal screening tests, such as maternal serum tests and ultrasound scan, have limited resolution and accuracy.
We developed an advanced noninvasive prenatal diagnosis method based on massively parallel sequencing. The Noninvasive Fetal Trisomy (NIFTY) test, combines an optimized Student’s t-test with a locally weighted polynomial regression and binary hypotheses. We applied the NIFTY test to 903 pregnancies and compared the diagnostic results with those of full karyotyping.
16 of 16 trisomy 21, 12 of 12 trisomy 18, two of two trisomy 13, three of four 45, X, one of one XYY and two of two XXY abnormalities were correctly identified. But one false positive case of trisomy 18 and one false negative case of 45, X were observed. The test performed with 100% sensitivity and 99.9% specificity for autosomal aneuploidies and 85.7% sensitivity and 99.9% specificity for sex chromosomal aneuploidies. Compared with three previously reported z-score approaches with/without GC-bias removal and with internal control, the NIFTY test was more accurate and robust for the detection of both autosomal and sex chromosomal aneuploidies in fetuses.
Our study demonstrates a powerful and reliable methodology for noninvasive prenatal diagnosis.
Noninvasive Fetal Trisomy (NIFTY) test; Massively parallel sequencing; Autosomal aneuploidies; Sex chromosomal aneuploidies
We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins.
Two dimensional capillary electrophoresis; ESI-MS; Magnetic beads
Years of education are inversely related to the prevalence of major depressive disorder (MDD), but the relationship between the clinical features of MDD and educational status is poorly understood. We investigated this in 1970 Chinese women with recurrent MDD identified in a clinical setting.
Clinical and demographic features were obtained from 1970 Han Chinese women with DSM-IV major depression between 30 and 60 years of age across China. Analysis of linear, logistic and multiple logistic regression models were used to determine the association between educational level and clinical features of MDD.
Subjects with more years of education are more likely to have MDD, with an odds ratio of 1.14 for those with more than ten years. Low educational status is not associated with an increase in the number of episodes, nor with increased rates of co-morbidity with anxiety disorders. Education impacts differentially on the symptoms of depression: lower educational attainment is associated with more biological symptoms and increased suicidal ideation and plans to commit suicide.
Findings may not generalize to males or to other patient populations. Since the threshold for treatment seeking differs as a function of education there may an ascertainment bias in the sample.
The relationship between symptoms of MDD and educational status in Chinese women is unexpectedly complex. Our findings are inconsistent with the simple hypothesis from European and US reports that low levels of educational attainment increase the risk and severity of MDD.
Major depressive disorder; Education; Socio-economic status; Symptom
Post partum depression (PPD) is relatively common in China but its clinical characteristics and risk factors have not been studied. We set out to investigate whether known risk factors for PPD could be found in Chinese women.
A case control design was used to determine the impact of known risk factors for PPD in a cohort of 1970 Chinese women with recurrent DSM-IV major depressive disorder (MDD). In a within-case design we examined the risk factors for PPD in patients with recurrent MDD. We compared the clinical features of MDD in cases with PPD to those without MDD. Odds ratios were calculated using logistic and ordinal regression.
Lower occupational and educational statuses increased the risk of PPD, as did a history of pre-menstrual symptoms, stressful life events and elevated levels of the personality trait of neuroticism. Patients with PPD and MDD were more likely to experience a comorbid anxiety disorder, had a younger age of onset of MDD, have higher levels of neuroticism and dysthymia.
Results obtained in this clinical sample may not be applicable to PPD within the community. Data were obtained retrospectively and we do not know whether the correlations we observe have the same causes as those operating in other populations.
Our results are consistent with the hypothesis that the despite cultural differences between Chinese and Western women, the phenomenology and risk factors for PPD are very similar.
Postpartum depression; Major depressive disorder; Neuroticism; Anxiety disorder
Diagnostic information for psychiatric research often depends on both clinical interviews and medical records. Although discrepancies between these two sources are well known, there have been few studies into the degree and origins of inconsistencies.
We compared data from structured interviews and medical records on 1,970 Han Chinese women with recurrent DSM-IV major depression (MD). Correlations were high for age at onset of MD (0.93) and number of episodes (0.70), intermediate for family history (+0.62) and duration of longest episode (+0.43) and variable but generally more modest for individual depressive symptoms (mean kappa = 0.32). Four factors were identified for twelve symptoms from medical records and the same four factors emerged from analysis of structured interviews. Factor congruencies were high but the correlation of factors between interviews and records were modest (i.e. +0.2 to +0.4).
Structured interviews and medical records are highly concordant for age of onset, and the number and length of episodes, but agree more modestly for individual symptoms and symptom factors. The modesty of these correlations probably arises from multiple factors including i) inconsistency in the definition of the worst episode, ii) inaccuracies in self-report and iii) difficulties in coding medical records where symptoms were recorded solely for clinical purposes.
The personality trait of neuroticism is a risk factor for major depressive disorder (MDD), but this relationship has not been demonstrated in clinical samples from Asia.
We examined a large-scale clinical study of Chinese Han women with recurrent major depression and community-acquired controls.
Elevated levels of neuroticism increased the risk for lifetime MDD (with an odds ratio of 1.37 per SD), contributed to the comorbidity of MDD with anxiety disorders, and predicted the onset and severity of MDD. Our findings largely replicate those obtained in clinical populations in Europe and US but differ in two ways: we did not find a relationship between melancholia and neuroticism; we found lower mean scores for neuroticism (3.6 in our community control sample).
Our findings do not apply to MDD in community-acquired samples and may be limited to Han Chinese women. It is not possible to determine whether the association between neuroticism and MDD reflects a causal relationship.
Neuroticism acts as a risk factor for MDD in Chinese women, as it does in the West and may particularly predispose to comorbidity with anxiety disorders. Cultural factors may have an important effect on its measurement.
Major depressive disorder; Anxiety disorders; Neuroticism
The relationship between major depressive disorder (MDD) and dysthymia, a form of chronic depression, is complex. The two conditions are highly comorbid and it is unclear whether they are two separate disease entities. We investigated the extent to which patients with dysthymia superimposed on major depression can be distinguished from those with recurrent MDD.
We examined the clinical features in 1970 Han Chinese women with MDD (DSM-IV) between 30 and 60 years of age across China. Logistic regression was used to determine the association between clinical features of MDD and dysthymia and between dysthymia and disorders comorbid with major depression.
The 354 cases with dysthymia had more severe MDD than those without, with more episodes of MDD and greater co-morbidity for anxiety disorders. Patients with dysthymia had higher neuroticism scores and were more likely to have a family history of MDD. They were also more likely to have suffered serious life events.
Results were obtained in a clinically ascertained sample of Chinese women and may not generalize to community-acquired samples or to other populations. It is not possible to determine whether the associations represent causal relationships.
The additional diagnosis of dysthymia in Chinese women with recurrent MDD defines a meaningful and potentially important subtype. We conclude that in some circumstances it is possible to distinguish double depression from recurrent MDD.
Major depressive disorder; Dysthymia; Symptom; Comorbidity
Individuals with early-onset depression may be a clinically distinct group with particular symptom patterns, illness course, comorbidity and family history. This question has not been previously investigated in a Han Chinese population.
We examined the clinical features of 1970 Han Chinese women with DSM-IV major depressive disorder (MDD) between 30 and 60 years of age across China. Analysis of linear, logistic and multiple logistic regression models was used to determine the association between age at onset (AAO) with continuous, binary and discrete characteristic clinical features of MDD.
Earlier AAO was associated with more suicidal ideation and attempts and higher neuroticism, but fewer sleep, appetite and weight changes. Patients with an earlier AAO were more likely to suffer a chronic course (longer illness duration, more MDD episodes and longer index episode), increased rates of MDD in their parents and a lower likelihood of marriage. They tend to have higher comorbidity with anxiety disorders (general anxiety disorder, social phobia and agoraphobia) and dysthymia.
Early AAO in MDD may be an index of a more severe, highly comorbid and familial disorder. Our findings indicate that the features of MDD in China are similar to those reported elsewhere in the world.
Major depressive disorder; Age at onset; Symptom; Comorbidity
In European and US studies, patients with major depressive disorder (MDD) report more stressful life events (SLEs) than controls, but this relationship has rarely been studied in Chinese populations.
Sixteen lifetime SLEs were assessed at interview in two groups of Han Chinese women: 1970 clinically ascertained with recurrent MDD and 2597 matched controls. Diagnostic and other risk factor information was assessed at personal interview. Odds ratios (ORs) were calculated by logistic regression.
60% of controls and 72% of cases reported at least one lifetime SLE. Fourteen of the sixteen SLEs occurred significantly more frequently in those with MDD (median odds ratio of 1.6). The three SLEs most strongly associated with risk for MDD (OR > 3.0) preceded the onset of MDD the majority of the time: rape (82%), physical abuse (100%) and serious neglect (99%).
Our results may apply to females only. SLEs were rated retrospectively and are subject to biases in recollection. We did not assess contextual information for each life event.
More severe SLEs are more strongly associated with MDD. These results support the involvement of psychosocial adversity in the etiology of MDD in China.
Major depressive disorder; Stressful life event; Social adversity; Symptom
A number of clinical features potentially reflect an individual's familial vulnerability to major depression (MD), including early age at onset, recurrence, impairment, episode duration, and the number and pattern of depressive symptoms. However, these results are drawn from studies that have exclusively examined individuals from a European ethnic background. We investigated which clinical features of depressive illness index familial vulnerability in Han Chinese females with MD.
We used lifetime MD and associated clinical features assessed at personal interview in 1,970 Han Chinese women with DSM-IV MD between 30–60 years of age. Odds Ratios were calculated by logistic regression.
Individuals with a high familial risk for MD are characterized by severe episodes of MD without known precipitants (such as stress life events) and are less likely to feel irritable/angry or anxious/nervous.
The association between family history of MD and the lack of a precipitating stressor, traditionally a characteristic of endogenous or biological depression, may reflect the association seen in other samples between recurrent MD and a positive family history. The symptomatic associations we have seen may reflect a familial predisposition to other dimensions of psychopathology, such as externalizing disorders or anxiety states. Depression and Anxiety 0:1–6, 2011. © 2011 Wiley-Liss, Inc.
major depression; family history; symptom; life events
Although the diagnosis of melancholia has had a long history, the validity of the current DSM-IV definition remains contentious. We report here the first detailed comparison of melancholic and nonmelancholic major depression (MD) in a Chinese population examining in particular whether these two forms of MD differ quantitatively or qualitatively.
DSM-IV criteria for melancholia were applied to 1,970 Han Chinese women with recurrent MD recruited from 53 provincial mental health centers and psychiatric departments of general medical hospitals in 41 cities. Statistical analyses, utilizing Student's t-tests and Pearson's χ2, were calculated using SPSS 13.0.
Melancholic patients with MD were distinguished from nonmelancholic by being older, having a later age at onset, more episodes of illness and meeting more A criteria. They also had higher levels of neuroticism and rates of lifetime generalized anxiety disorder, panic disorder, and social and agoraphobia. They had significantly lower rates of childhood sexual abuse but did not differ on other stressful life events or rates of MD in their families.
Consistent with most prior findings in European and US populations, we find that melancholia is a more clinically severe syndrome than nonmelancholic depression with higher rates of comorbidity. The evidence that it is a more “biological” or qualitatively distinct syndrome, however, is mixed.
major depression; melancholia; symptom; stressful life events
The relationship between recurrent major depression (MD) in women and suicidality is complex. We investigated the extent to which patients who suffered with various forms of suicidal symptomatology can be distinguished from those subjects without such symptoms.
We examined the clinical features of the worst episode in 1970 Han Chinese women with recurrent DSM-IV MD between the ages of 30 and 60 years from across China. Student's t tests, and logistic and multiple logistic regression models were used to determine the association between suicidality and other clinical features of MD.
Suicidal symptomatology is significantly associated with a more severe form of MD, as indexed by both the number of episodes and number of MD symptoms. Patients reporting suicidal thoughts, plans or attempts experienced a significantly greater number of stressful life events. The depressive symptom most strongly associated with lifetime suicide attempt was feelings of worthlessness (odds ratio 4.25, 95% confidence interval 2.9–6.3). Excessive guilt, diminished concentration and impaired decision-making were also significantly associated with a suicide attempt.
This study contributes to the existing literature on risk factors for suicidal symptomatology in depressed women. Identifying specific depressive symptoms and co-morbid psychiatric disorders may help improve the clinical assessment of suicide risk in depressed patients. These findings could be helpful in identifying those who need more intense treatment strategies in order to prevent suicide.
Co-morbidity; major depression; suicidal ideation; suicide; women