• LMW peptides
• MALDI−MS
• non-cryopreservation exposure
• preanalytical factors
• protease

• Female
• Humans
• Breast Neoplasms/diagnosis
• Breast Neoplasms/epidemiology
• Longitudinal Studies
• Mass Screening/methods
• Prospective Studies
• Risk Factors
• Multicenter Studies as Topic

• batches
• blocking
• experimental design
• randomization
• sample handling

• Proteomics
• Random Allocation
• Research Design

• AHSG
• APOA-I
• Biomarker
• FGA
• Gastric cancer

Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. Plasma test samples from the – 80 °C freezer were thawed on ice or intentionally warmed to room temperature.

Protein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) and correlated with X!TANDEM.

Fully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than “no enzyme” correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours–days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra.

The peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.

The online version of this article (10.1186/s12014-017-9176-7) contains supplementary material, which is available to authorized users.

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptides from albumin, complement, and apolipoproteins and others that were observed by the X!TANDEM and SEQUEST algorithms. In contrast to ice cold samples, after incubation at room temperature, greater numbers of tryptic peptides from well characterized plasma proteins, and from cellular proteins were observed. A total of 583,927 precursor ions and MS/MS spectra were correlated to 94,669 best fit peptides that reduced to 22,287 correlations to the best accession within a gene symbol and to 7174 correlations to at least 510 gene symbols with ≥ 5 independent MS/MS correlations (peptide counts) that showed FDR q-values ranging from E−9 (i.e. FDR = 0.000000001) to E−227. A set of 528 gene symbols identified by X!TANDEM and SEQUEST including C4B showed ≥ fivefold variation between ice cold versus room temperature incubation. STRING analysis of the protein gene symbols observed from endogenous peptides in normal plasma revealed an extensive protein-interaction network of cellular factors associated with cell signalling and regulation, the formation of membrane bound organelles, cellular exosomes and exocytosis network proteins. Taken together the results indicated that a pool of cellular proteins, or protein complexes, in plasma are apparently not stable and degrade soon after incubation at room temperature.

• Big Data
• blood-derived biospecimens
• multi-omics
• preanalytical variation
• precision biobanking

• Pre-analytical variability
• sample handling
• sample matrix
• stability

High quality clinical samples are critical for meaningful interpretation of data obtained in both basic and translational medicine. More specifically, optimized pre-analysis handling to bio-sample is crucial for avoiding biased analysis in a clinical setting. A universally applicable method for the evaluation of sample quality and pre-analysis handling is therefore in great demand.

The fingerprint pattern of low molecular weight (LMW) peptides in sera is directly associated with sample quality and handling process. Previous studies for enrichment/isolation of LMW peptides have shown that LMW peptides can be enriched by silica meso-porous material in a sensitive and high-throughput manner. Here, a peptide profile approach utilizing mesoporous silica chip-based sample preparation combined with MALDI MS analysis was used as a new platform for evaluation of bio-sample quality. Rat sera were selected as model sample and analyzed according to their LMW peptide fingerprint spectra.

This novel method can complete the entire sample preparation procedure in a short period of time (<40 min), requires minimum amounts of sample (<10 µL), is of high sensitivity (LOD 10 ng/mL) as well as high reproducibility (CV% < 15%). According to the acquired LMW peptide spectra, we were able to distinguish the serum samples processed under different conditions (including different storage temperature, time, and freezing/thaw cycles) with the help of bioinformatics tools (principle composition analysis and significant difference analysis), and identify the samples that had significantly changed due to the inappropriate processing. Based on the percentage of significantly changed peaks in LMW peptide mass spectrum after handling, a judgment standard was established that can be used to evaluate the status of preservation of a biological sample. In addition, our principle study established recommendations for storage time, storage temperature and freeze/thaw conditions.

Our novel method for analysis of bio-samples allows for effective identification of variations in composition within samples, and provides a cost-effective tool for simple sample manipulation in a clinical setting.

• LMW peptide
• MALDI MS
• Pre-analysis handling
• Sample quality
• Silica mesoporous material

• Apolipoprotein C-I
• biomarker
• breast cancer
• diagnosis
• mass spectrometry
• prognosis
• serum

Gastric cancer (GC) is a highly aggressive cancer type associated with significant mortality owing to delayed diagnosis and non-specific symptoms observed in the early stages. Therefore, identification of novel specific GC serum biomarkers for screening purposes is an urgent clinical requirement.

This study recruited a total of 432 serum samples from 296 GC patients split into the mining and testing sets. We aimed to screen for reliable protein biomarkers from matched serum samples based on mass spectrometry, followed by comparison with three representative conventional markers using receiver operating characteristic and survival curve analyses to ascertain their potential values as diagnostic and prognostic biomarkers for GC.

We identified an apoC-III fragment with confirmation in an independent test set from a second hospital. We found that the diagnostic ability of this fragment performed better than current standard GC diagnostic biomarkers both individually and in combination in distinguishing patients with GC from healthy individuals. Moreover, we found that this apoC-III protein fragment represents a more robust potential prognostic factor for GC than the three conventional markers.

In view of these findings, we suggest that apoC-III protein fragment is a novel diagnostic and prognostic biomarker, a complement to conventional biomarkers in detecting GC.

• SELDI-TOF MS
• biomarker
• breast cancer
• fractionation
• proteomics

Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system. The time frame for development from premalignant to malignant disease typically spans 10-15 years, and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes. Currently, early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for population-based screening. New blood-based protein biomarkers for early detection of CRC are therefore urgently required. The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental, analytical, and biological factors that can interfere with the robust interpretation of results. In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies. Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.

• Biomarker
• Bias
• Colorectal cancer
• Diagnostic
• Discovery
• Validation

• Animals
• Biomarkers, Tumor/blood
• Colorectal Neoplasms/blood
• Colorectal Neoplasms/mortality
• Colorectal Neoplasms/pathology
• Colorectal Neoplasms/therapy
• Early Detection of Cancer
• Humans
• Predictive Value of Tests
• Prognosis
• Reagent Kits, Diagnostic
• Reproducibility of Results
• Specimen Handling
• Time Factors

Noninvasive biomarkers are urgently needed for early detection of breast cancer since the risk of recurrence, morbidity and mortality are closely related to disease stage at the time of primary surgery. In the past decade, many proteomics-based approaches were developed that utilize the protein profiling of human body fluids or identification of putative biomarkers to obtain more knowledge on the effects of cancer emergence and progression. Herein, we report on an analysis of proteins in the tear fluid from breast carcinoma patients and healthy women using a de novo proteomic approach and 25 mixed samples from each group. This study included 25 patients with primary invasive breast carcinoma and 25 age-matched healthy controls. We performed a MALDI-TOF-TOF-driven semi-quantitative comparison of tear protein levels in cancer (CA) and control (CTRL) using a de novo approach in pooled samples. Over 150 proteins in the tear fluid of CTRL and CA were identified. Using an in-house-developed algorithm we found more than 20 proteins distinctly upregulated or downregulated in the CTRL and CA groups. We identified several proteins that had modified expression in breast cancer patients. These proteins are involved in host immune system pathways (e.g., C1Q1 or S100A8) and different metabolic cascades (ALDH3A or TPI). Further validation of the results in an independent population combined with individual protein profiling of participants is needed to confirm the specificity of our findings and may lead to a better understanding of the pathological mechanism of breast cancer.

• BLAST
• ESI
• HPLC
• LC-MS/MS
• MALDI
• MS MS
• MS-MS
• MS/MS
• PAGE
• SEQUID
• SQL
• UPLC
• analysis
• bioinformatics
• blood, serum
• chromatography
• circulating
• circulation
• database
• electrophoresis
• electrospray ionization
• ion
• liquid chromatography mass spectrometry
• mass spectra
• mass spectrom
• mass spectrometry
• mass spectrum
• matrix assisted laser desorption ionization
• peptide
• peptidome
• peptidomics
• plasma
• protein
• proteome
• proteomics
• sera
• statistics
• tandem mass spectrometry

• Blood Proteins/isolation & purification
• Blood Proteins/metabolism
• Databases, Protein
• Endopeptidases/chemistry
• Humans
• Mass Spectrometry/instrumentation
• Mass Spectrometry/methods
• Protein Processing, Post-Translational
• Proteolysis
• Reference Standards
• Sensitivity and Specificity
• Specimen Handling

Serum protein profiles have been investigated frequently to discover early biomarkers for breast cancer. So far, these studies used biological samples collected at or after diagnosis. This may limit these studies' value in the search for cancer biomarkers because of the often advanced tumor stage, and consequently risk of reverse causality. We present for the first time pre-diagnostic serum protein profiles in relation to breast cancer, using the Prospect-EPIC (European Prospective Investigation into Cancer and nutrition) cohort.

In a nested case-control design we compared 68 women diagnosed with breast cancer within three years after enrollment, with 68 matched controls for differences in serum protein profiles. All samples were analyzed with SELDI-TOF MS (surface enhanced laser desorption/ionization time-of-flight mass spectrometry). In a subset of 20 case-control pairs, the serum proteome was identified and relatively quantified using isobaric Tags for Relative and Absolute Quantification (iTRAQ) and online two-dimensional nano-liquid chromatography coupled with tandem MS (2D-nanoLC-MS/MS).

Two SELDI-TOF MS peaks with m/z 3323 and 8939, which probably represent doubly charged apolipoprotein C-I and C3a des-arginine anaphylatoxin (C3a_desArg), were higher in pre-diagnostic breast cancer serum (p = 0.02 and p = 0.06, respectively). With 2D-nanoLC-MS/MS, afamin, apolipoprotein E and isoform 1 of inter-alpha trypsin inhibitor heavy chain H4 (ITIH4) were found to be higher in pre-diagnostic breast cancer (p < 0.05), while alpha-2-macroglobulin and ceruloplasmin were lower (p < 0.05). C3a_desArg and ITIH4 have previously been related to the presence of symptomatic and/or mammographically detectable breast cancer.

We show that serum protein profiles are already altered up to three years before breast cancer detection.